Energy & Fuels 1995,9, 155-162
155
Application of Biological Marker Technology to Bioremediation of Refinery By-Products J. Michael Moldowan,*r?>lJeremy Dahl,? Mark A. McCaffrey,t William J. Smith,$ and John C. Fetzer8 Chevron Petroleum Technology Company, P.O. Box 1627, Richmond, California 94802-0627, and P.O. Box 446, La Habra, California 90633-0446; Chevron Research and Technology Company, P.O. Box 1627, Richmond, California 94802-0627 Received April 4, 1994. Revised Manuscript Received September 26, 1994@
The progress of bioremediation of waste petroleum sludge a t Chevron’s Perth Amboy, New Jersey, refinery landfarm was evaluated using a ranking scale based on refractory biological marker hydrocarbons that are indigenous to, and ubiquitous in, crude oils. Of the four samples analyzed from different locations in the landfarm, two were virtually identical and showed an absence of the n-alkanes expected to be found in the sludge (light biodegradation ranking). Another showed additional partial degradation of acyclic isoprenoids, e.g., pristane and phytane (moderate ranking). The fourth sample showed complete n-paraffin and isoprenoid loss, partial alteration of hopanes, and losses of C27 steranes, C27 diasteranes, C27 monoaromatic steroids, and c 2 6 triaromatic steroids relative to the higher steroid homologs in each of these series (heavy ranking). These results suggest a concomitant preferential loss of steroid hydrocarbons that have the cholestane side chain and a possible new steroid biodegradation mechanism that is essentially blind to the structure of the steroid nucleus. The latter sample also showed levels of most polynuclear aromatic hydrocarbons (PAH), suggesting a buildup of these compounds as others were removed. However, some of the smaller PAH (acenaphylene, fluorene, fluoranthene) appear to have decreased. These results suggest that a protocol based on such a biodegradation ranking scale could be used to monitor the progress of bioremediation of oil based refinery wastes.
Introduction During production and refining operations, numerous opportunities exist for crude oil and oil products to enter the environment. Although oil spills can have significant environmental impact, the methods typically applied for monitoring microbial degradation of these petroleum mixtures are only qualitative to semiquantitative at best. Methods that have been employed include (1) simple extraction and gravimetric or IR analysis of oily substances, sometimes assisted by gas chromatographic analysis; (2) indirect estimation of the hydrocarbon transformed from the amount of oxygen consumed or carbon dioxide produced; and (3) analysis of individual constituents of petroleum, especially volatile constituents like benzene, toluene, ethylbenzene, and xylenes (BTEX) and semivolatile constituents like naphthalene and other multiring polycyclic aromatic compounds. Because these compounds are produced by nonpetroleum sources, interpretations of analytical results may be plagued by uncertainties about their origin. In addition to petroleum products, the extraction method picks up many hydrocarbons and other lipids originating from microorganisms, plants, and animals that live in or on the soil. Microorganisms consume oxygen and produce carbon dioxide when they grow on either hydrocarbons or non-hydrocarbon substrates; +Chevron Petroleum Technology Co. P.O. Box 1627. Chevron Petroleum Technology Co. P.O. Box 446. Chevron Research and Technology Co., P.O.Box 1627. Current address: Stanford University, Department of Geological and Environmental Sciences, Stanford, CA 94305-2115. Abstract published in Advance ACS Abstracts, November 1,1994.
* *
@
0887-0624/95/2509-0155$09.00/0
furthermore, these organisms have metabolic pathways available that differ by a factor of 2 or more in the amount of oxygen consumed. Therefore, indirect estimation methods based on oxygen consumption or carbon dioxide production are difficult t o interpret, especially outside of the laboratory environment. Since individual constituents differ greatly both in the amount initially present within the petroleum fraction and in their rates of biodegradation, monitoring easily degradable constituents tells very little about the fate of the thousands of other more refractory constituents typically found in petroleum. This work directly measures the relative concentrations of several of a special class of refractory hydrocarbons, biomarkers. Biomarkers, or molecular fossils, are compounds that can be related by their structures to biological precursors. They are ubiquitous in crude oils and survive many refinery processes, ending up in the medium to heavy fracti0ns.l Petroleum biomarker fingerprints are distinctive, and they cannot originate from other sources. Whereas the fate of easily degradable constituents provides little information on the fate of refractory hydrocarbons, the disappearance of refractory hydrocarbons, for example biomarkers, may indicate that most, if not all, of the more easily degradable constituents have been transformed. This suggests that it may be possible t o adapt a scale for ranking the biodegradation of crude oils to monitoring the bioremediation of hun(Figure 1)2,3 (1)Peters, K.E.;Scheuerman, G. L.; Moldowan, J. M.; Lee, C. Y.; Reynolds, R. N.; Pefia, M. M. Energy Fuels 1992,6,560-577.
0 1995 American Chemical Society
156 Energy & Fuels,
Moldowan et al.
Vol. 9,No. 1, 1995
Extent of Destructionof Compound Class 1
Light
1
I I
1
I 2
3 Moderate
I I I I I
I I I I
5
H“sw
1
I I
(6)
I 7 very p
w
Soil Profile at Landfarm Prior to Waste Application
Ammatla Hopones D I ~ t e n n w c&,
Blodsgrsdetlon Ranklng ~PorcltllnaIsoprenolds Stennes
1 1 I I
I I
I I I I I I I I I I I I I I
I I
I I
Soil Surface
I I I I I I I I
I I I I I
I I I I I I 1
Synthetic Membrane Liner
9
Severe 10
1 s Lamhomolog.ofnp.mfflnr&plotul. 2 t ci-I apktlon of n-pmnfflno. 3 = Only t”of n-pmWlnr remln. 4 = No n-prnfilnr, q c l l c IsoprartoldaI-. 5 t Acycllc lsoprenoldr absent.
6 . StnmrpmVdrgndrd. 7a St.mr&glSd.d,d*~m(KI. 8 t H o p s m p r t l y d0g”J. 9 zz Hopawa sbaml. dlasttarm .mclud 10 t C X C a rromatk rtbloldr .ttrx.d.
Figure 1. Biological marker biodegradation ranking chart based on the natural biodegradation of crude oils.3
dreds of millions of cubic yards of soil contaminated with refined petroleum products and wastes. Biomarkers have recently been introduced for monitoring remediation effectiveness. Following the Exxon Valdez oil spill in 1989, the fate of deposits at various contaminated sites in Prince William Sound was monitored using hopane, a biodegradation-resistant biomarker found in the 0i1.~-~Because no measurable degradation of hopane was found, it was considered invaluable as an “internal standard” to calculate losses of other hydrocarbons from the original oil. The data of the EXXON gr0up5p6suggest that biomarker biodegradation had not occurred in the oil spilled from the EXXON Valdez. In contrast, numerous studies of naturally occurring seepage oils and shallow reservoired crude have shown that biomarker biodegradation, including hopane, is very ~ o m m o n . ~The . ~ relationship between biodegradation of natural seeps, oil spills, petroleum waste disposal projects, and the effects on biomarkers needs to be systematically studied to clarify the ongoing applications of this technology. Biodegradation of oil in soils occurs under different conditions than in reservoirs (e.g., oxygen, nutrients, temperature), and one might expect some differences in the sequence (Figure 1)of biomarker biodegradation. If all the known toxic compounds degrade before certain biomarkers, a biomarker based biodegradation ranking (2)Moldowan, J. M.; Lee, C. Y.; Sundararaman, P.; Salvatori, R.; Alajbeg, A.; Gjukic, B.; Demaison, G. J.; Slougui, N. E.; Watt, D. S. Biological Markers in Sediments and Petroleum; Moldowan, J. M., Albrecht, P., Philp, R. P., Eds.; Prentice Hall Publishing Co.: Englewood Cliffs, NJ, 1992;p 370-401. (3)Peters, K. E.;Moldowan, J. M. The Biomarker Guide; Prentice Hall Publishing Company: Englewood Cliffs, NJ, 1993;363 pp. (4)Bragg, J. R.; Prince, R. C.; Harner, E. J.; Atlas, R. M. Prepr.-Diu. 239. Pet. Chem. 1993,38(2), (5) Prince, R. C., Elmendorf, D. L., Lute, J. R.; Hsu, C. S.; Halth, C. E.; Senius, J. D.; Dechert, G. J.; Douglas, G. S.; Butler, E. L. Enuiron. Sci. Technol. 1994,28,142-145. (6)Prince, R.C.; Hinton, S. M.; Bragg, J. R.; Elmendorf, D. L.; Lute, J. R.; Grossman, M. J.; Robbins, W. K.; Hsu, C. S.; Douglas, G. S.; Bare, R. E.; Haith, C. E.; Senius, J. D.; Minak-Bernero, V.; McMillan, S. J.; Roffall, J. C.; Vhianelli, R. R. Prepr.-Diu. Pet. Chem. 1993,38(2), 240-244. (7)Chosson, P.; Connan, J.; Dessort, D.; Lanau, C. Biological Markers in Sediments and Petroleum; Moldowan, J. M., et al., Eds.; Prentice Hall Publishing Co.: Englewood Cliffs, NJ, 1992;pp 320349.
-_ _
~
Figure 2. Stratigraphy of the Perth Amboy landfarm.
scale could prove to be an absolute one that could be used to evaluate the efficacy of bioremediation efforts. Its application might eventually be used to answer for a variety of hydrocarbons the presently unanswerable question, “How clean is clean?”, a question which impedes regulatory acceptance of bioremediation. To test the applicability of biomarker distributions as a tool for monitoring bioremediation of refinery wastes, we analyzed the biomarkers in four soil samples from the Perth Amboy landfarm at the Chevron Perth Amboy Refinery, in Perth Amboy, New Jersey. This is the first study that attempts to determine whether biomarkers in refinery wastes can be expected to biodegrade at all under landfarm conditions.
Experimental Section Landfarm Description and Sampling. Between August of 1981 and October of 1982 approximately 600 tons of waste sludge from API separators, induced air flotation units, and non-leaded tank bottoms were applied to the Perth Amboy landfarm. Petroleum sludge such as this typically contains at most a few hundred ppm volatile (e.g., benzene, toluene, ethylbenzene, and xylene) and semivolatile hydrocarbons. This sludge consisted primarily of higher molecular weight, nonvolatile hydrocarbons, including asphaltenes. The top layer of the landfarm is a heterogeneous mixture of sand and soil approximately 18in. thick (Figure 2). Borings taken in December of 1984 showed the uniformity with which oil and grease had been applied across the surface of the landfarm. Oil and grease concentrations in these borings from three corners and the center of the landfarm ranged from 2.1 to 5.1 wt % hydrocarbon, or, in other words, a 2.5-fold range from lowest to highest. No sample was taken from the northwest corner. The landfarm was actively tilled during the 14 months waste was applied to it. Active tilling ceased in October of 1982 and then resumed during the summer of 1990. When tilling resumed in 1990, bioremediation in the southern section of the landfarm had proceeded more rapidly than that in the northern section, especially the northeast corner. The landfarm drained toward the northeast corner, and the soil in this corner is saturated with water throughout most of the year. Slow oxygen diffusion rates limit the rate of bioremediation in saturated soils. Four soil samples were collected in March 1991 from the top 12 in. of soil of the landfarm. The sample from the northeast corner (A828) had a strong hydrocarbon odor and
Energy & Fuels, Vol. 9, No. 1, 1995 157
Bioremediation of Refinery By-products
(b) Middle Landfarm
(a) North End Landfarm
Sample No. A829
Sample No. A828
Pristane
\
,y
Phytane
I Prisy 1 Phytane
(c) Middle Landfarm
(d) South End Landfarm
Sample No. A830
Sample No. A831
Phvtane
=
i
Hopanes
IprisY I
lsoprenoidr Absent
20
-
50
80
I
UCM Degraded
110
GC T h e Dlrection
20
50
80
110
GC T h e Direction
Figure 3. Gas chromatography-mass spectrometry total ion current chromatograms for four landfarm samples, saturate fractions. free oil and water were visible a few inches below the surface. Two samples from the central (A8291 and north central (A830) and one from the southern (A831) sections of the landfarm were friable, free of hydrocarbon odors, and exhibited no visual evidence of hydrocarbons. After March 1991 the farm was fertilized and tilled, and in October 1991 three additional samples were collected from a single core in the southeast corner. The three samples were taken from the top 2 cm of soil, from 3-4 cm depth, and from 8-9 cm depth. Sample Analysis. For each soil sample, approximately 30 g of moist sample was sonicated in 200 mL of methanol for 10 min followed by centrifugation at 1800 rpm for 20 min. The supernatant was decanted into a separatory funnel, and extracted three times, each with 100 mL of hexane. The MeOH extracted soil was then extracted using a Soxtec for 4 h in Me0H:toluene (2:l) followed by 3 h in CHC13:MeOH (87: 13). The Soxtec extracts were combined with the hexane extracts, rotoevaporated t o remove solvents, and weighed. Asphaltenes and some polar material were then removed by passing the sample through an alumina gravity column in 90% hexane, 10% tert-butyl ether. The samples were then separated into saturate, aromatic, and polar fractions by HPLC using the method of Peters and M ~ l d o w a n .The ~ saturate fraction was analyzed by GC-MS on a VG Trio-1 mass spectrometer interfaced with a Hewlett Packard 5890 gas chromatograph. The GC temperature program was isothermal at 140 "C for 5 min, 2 "C/min t o 320 "C, and isothermal at 320 "C for 20 min, using Hz as the carrier gas. Transfer line temperature was 320 "C.Injection was splitless at 325 "C and the column a 60 m J&W DB-1 (0.25 pm film thickness, 0.25 mm i.d.1. The spectrometer was run in full scan mode, 1s c a d 1.5 s using E1 at 70 eV as the ionization mode. To quantify the steranes, the saturate fraction was spiked with 5B-cholane and analyzed using metastable reaction monitoring (MRM) on
a VG 7070H mass spectrometer. Response factors for each sterane were determined from a standard oil that contains a known quantity of each sterane and which was also spiked with 5B-ch0lane.~ PAH analysis was limited to parent compounds, which are most commonly found on regulatory lists. Standards of 27 PAH's (from Bureau of Community Reference, Brussels; CTC Organics, Atlanta; Aldrich Chemical Co., Milwaukee) were prepared. Solvents were HPLC-grade acetonitrile, dichloromethane, and water (Burdick and Jackson, Muskegon). An internal recovery standard, perdeuteroperylene (ICN Biochemicals, Cambridge), was added to the soil samples before extraction. Standard reference material 1647 (National Institute for Standards and Technology, Gaithersburg, MD) was also used as a quantitation standard. Each sample was dissolved in 5 mL of dichloromethane and diluted with 15 mL of acetonitrile. A Perkin-Elmer Model 410 quaternary solvent HPLC with a 250 pL loop injector and a dual-monochromator fluorescence detector (Shimadzu RF-530) was used for aromatic compound analysis. The column was a Vydac 201TP5 (2-18 column (Separations Group, Hesperia, CA), 0.46 cm i.d. x 25 cm. The linear mobile phase gradient was 4455% acetonitrile/water t o 100% acetonitrile over 30 minutes at 1.50 m l / m h s Each compound was monitored with a retention time window fl min compared to standards. Wavelengths for the monochromators were chosen from reference s p e ~ t r a . ~There J~ were two sets of PAH's, each comprising the compounds (8) Sander, L. C.;Wise, S.A. Advances in Chromatography; Giddings, J. C., et al., Eds.; M. Dekker: New York, 1986; Vol. 25, pp 139218. (9) Berlman, I. M.Handbook of Fluorescence Spectra of Aromatic Compounds; Academic Press: New York, 1971. (10) Karcher, W. Spectral AtZm of Polycyclic Aromatic Compounds; Reidel Publishing: Boston, 1991; Vol. 1-3.
158 Energy & Fuels, Vol. 9,No. 1, 1995
Moldowan et al.
Chart 1. Biomarker Structures Mentioned in Text
Diasteraner. Cp. C30 R = H. LX3, G H , . nC,H7
R = 11. CH,.CqHs. n.C,H,
R
R
Tnaromatic steroids. R = H. C H 3 . C2HJ Monoaromatic steroids. CZ7.C , R = H. CH3. C?H!
1 60
-
80
70
GC Time Direction
’ ..
,
Bacteriohopanerenol (a likclv precursor of scncs 6)
@:::. ‘
9
(C-Ts)
Norncnhopanc
* 11. Pristane
’
olcanancs
Figure 4. Gas chromatography-mass spectrometry m l z 217 mass chromatograms for the saturate fractions of landfarm samples (a)A828 and (b)A831. Samples A828, A829, and A830 were nearly identical in this analysis. (0)
North End Landform A828
u
C&2SMR
IO. Norpristanc
= 1.5
* 12. Phytane
analyzable in one run. The wavelength reproducibility was 0.25-0.5 nm, so minimal differences in quantitation were found. The PAH analyses were repeated three times, with relative standard deviations of 7-8%. Although most PAH’s can be detected without wavelength switching, detection sensitivity and selectivity were considered to be major criterion. Since there was no background fluorescence and light scattering was minimal, the baseline shifted only slightly.
c
(b) South End Landform
80
90
I
100
Mln.
GC flme Dlmctlon
Results and Discussion Gas Chromatography-Mass Spectrometry, Total Ion Chromatograms. Gas chromatography-mass spectrometry (GC-MS) total ion chromatograms (TIC) of the saturate fractions of the four samples (Figure 3) show the absence of a regular n-alkane pattern. The three least biodegraded samples (A828-A830) contain the isoprenoids norpristane, pristane, and phytane (structures 10,11,and 12,respectively; see Chart 1for structures). The two northernmost samples (A828 and A829, Figure 3, a and b) differ slightly in their internal ratio of pristane and phytane, but otherwise appear identical. Some peaks in the 70-100 min elution range of these samples are hopane hydrocarbons (5 and 6). Partial removal of the acyclic isoprenoids in sample A830 (Figure 3c), and complete removal in the southernmost sample, A831, compared to samples A828 and A829, suggest an order of increasing biodegradation toward the south of the field. Figure 3d is scaled so that the C35 hopane (6)peaks are of similar height to the same peaks in Figure 3a-c. When the figure is scaled in this manner, it becomes clear that a portion of the unresolved complex mixture (UCM) has also been degraded in this sample. It is important to note that the apparent increase in the abundance of the steranes in this sample may actually be the result of degradation of the surrounding UCM.
Figure 5. Gas chromatography-mass spectrometry m / z 191 mass chromatograms for the saturate fractions of landfarm samples (a)A828 and (b)A83 1.Samples A828, A829, and A830 were nearly identical in this analysis.
The reduced abundance of the UCM relative to the steranes and the C35 hopanes probably provides a conservative estimate of the UCM degradation, since the C35 hopanes and steranes may have also been reduced in intensity by degradation. A substantial fraction of the original UCM in sample A831 has not been degraded. The composition of this material is unclear, although monoalkyl-substituted T-branched alkanes and cyclic alkanes have been suggested as the dominant compound classes in the UCM of biodegraded oils.11 Selected Ion Chromatography GC-MS and GCMS-MS. The general appearance of the mlz 217 and 191 mass chromatograms of the least biodegraded samples (A828-A830) is the same; they show sterane and terpane patterns (Figures 4 and 5, respectively) which are grossly similar to many crude oils. The distribution of C29 sterane isomers (1)contains 5a,14a, 17a(H),20R,24-ethylcholestane,the hydrocarbon with the same stereochemistry as the biologically produced sterols. In addition, those 24-ethylcholestanesproduced (11)Gough, M.A.; Rowland, S.J. Nature 1990,344,648-650.
Bioremediation of Refinery By-products
Energy &Fuels, Vol. 9, No. I, 1995 159
by geological processes, which include 5a,14a,17a(H), 20S, 5a,14/3,17/3(H),20S,and 5a,14/3,17p(H),20R (abbreviated sad, aaaS, apPS, and @/3R, respectively) are also present. The 13/3,17a(H)-diasteranes (2; e.g., c 2 7 diasteranes, Figure 4) are present as well. Diasteranes are formed geochemically in a series of steps including an acid (clay mineral)-catalyzed rearrangement during early diagenesis to form A13J7-diasterenes, followed by a hydrogenation of the double bond after burial of the sediment.12 Thus, the presence of the geologically produced sterane and diasterane isomers is indicative of crude oil constituents rather than a material emplaced by recent biological activities at the landfarm site. This conclusion is also supported by the presence of a full distribution of 17a,21/3(H)-hopanes(c29-c36, 5 and 6 ) which are formed by geological isomerization of 17/3,21/3(H)-hopanes. The c31-c36 a#Lhomohopanes(6) are equilibrated at (2-22 [22S/(22S+22R) FG 0.61 which identifies the analyte as an ancient, thermally-mature fossil fuel material.3 In contrast, 17/3,21/3(H)-hopanes and 17/3,21/3(H),22R-homohopanes(e.g., 7) with stereochemistries produced biochemically by prokaryotes13 are absent here and in virtually all petroleum. Geochemical Interpretation. Some information on the paleogenetic origin of the crude oil(s)can be gleaned from the biomarker patterns, for example, using the least degraded sample (A828). While the origin of this material was known by the refiners, that information was not available during the time of this study. This crude, or blend of crudes, apparently originated from a marine source rock(s) as evidenced by abundant C30 steranes (24-n-propylcholestanes,l), analyzed using metastable reaction monitoring (MRM) GC-MS of the m/z 414 to 217 metastable tran~iti0n.l~ Marine origin is further supported by a C35 homohopane (22s 2%) ( 6 ) doublet which is slightly predominant over that for the C34 homohopanes (Figure 5a). This condition is indicative of crude oil sourced from rocks deposited in a marine, anoxic basin.2 This anoxic marine condition is supported by a pristane/phytane ratio
