Environ. Sci. Techno/. 1995, 29, 1615-1621
Microbial Degradation in Soil Microcosms of Fuel Oil Hydrocarbons from Drilling Cuttings CLAUDE-HENRI CHAINEAU,' JEAN-LOUIS MOREL,' AND JEAN OUDOT*s' Ecole Nationale Supbrieure d'tlgronomie et des Industries Alimentaires, Laboratoire Sol et Enuironnement, 2 Auenue de la Fort2 de Haye, B.P. 172, 54505 Vandoeuure-Les-Nancy,Cedex, France, Musdum National d'Histoire Naturelle, Laboratoire de Cryptogamie, 12 rue Buffoon, 75005 Paris, France
The biodegradation of the fuel oil hydrocarbons contained in drilling cuttings was studied in soil microcosms during a 270-day experiment. Concentration and chemical composition of residual hydrocarbons were periodically monitored by quantitative capillary gas chromatography. The decrease in hydrocarbon concentration was logarithmic with time. A t the end of the experiment, the fuel oil was 75% degraded. In the saturated fraction, normal and branched alkanes were almost totally eliminated in 16 days; 22% of the cycloalkanes were not assimilated. The aromatic fraction was 71% degraded; some polycyclic aromatics were persistent. The resin fraction (10% of the initial weight) was completely refractory to biodegradation. The inorganic part of drilling cuttings had no influence on the biodegradation rates of hydrocarbons. Biogenic hydrocarbons and traces of degradable fuel oil hydrocarbons were protected from microbial activity by the soil and cuttings matrix. Enumerations of total heterotrophic bacteria and hydrocarbon-utilizing bacteria showed a strong stimulation in both po puIations. Hydro c a r bo n-degrad ing strains of bacteria and fungi were isolated and identified at the generic or specific level.
Introduction Onshore drilling operations generate oil-based wastes, typically 100-150 m3/well.The drilling cuttings consist of the drilling fluid (e.g.,hydrocarbons,water, additives) mixed with the subsurface material (inorganic matter). These wastes are disposed of in a variety of methods like incineration and controlled dumping. The landfarming or land treatment has been considered as a method to deal with oily wastes ( 1 - 5 ) . It has been well-documented that hydrocarbons (HC) are prone to be biodegraded by the microbial population of the soil environment (6-81,but the effects of the mineral components of drilling cuttings had not yet been studied. Since many wells are drilled in agricultural areas, the landfarming of drilling cuttings may represent an economically means for treating HC associated with drilling cuttings. However, it has alreadybeen pointed out that some petroleum compounds are resistant to biodegradation (9, 10). This work was undertaken during a 9-month experiment in the laboratory (i) to evaluate under controlled conditions the biodegradation of the HC of drilling cuttings incorporated in an agricultural soil and (ii) to measure their influence on the soil microbial community.
Experimental Methods Soil Microcosms. Soil was collected from an agricultural area with no history of hydrocarbon disposal but where drilling activities are important (Marne, France). The Ap horizon of an elluviated brown soil (typical hapludalfl was sampled, air-dried, and sieved (2-mm diameter openings) before analysis. It exhibited a silty-clay texture, had a pH of 5.1, and was rather high in P (Table 1). Drilling cuttings were obtained during drilling operations and kept at 4 OC before experiments; their composition is given in Table 1. The initial drilling fluid was an oil-based fluid composed of 66% fuel oil, 22% water, 6% CaC12,1.3%emulsifier, 1.7% fdtrate-reducing agent, and 3% Ca(OH)2. A series of 60 100-cm3 beakers was prepared, each containing 70 g of soil. Thirty-six were contaminated by adding and mixing 3.2 g of drilling cuttings in each beaker to obtain 2670pg (g of soil of fuel oil HC1-l (treated soils). The loading rate was relatively low; it corresponded to a ratio of 7 t /ha of fuel oil HC, which was shown in preliminary studies to have little impact on plant growth and to permit high degradation rates. Twelve beakers received the same amount of HC and were poisoned with 1% HgC12 (sterile soils). One percent HgC12 was added monthly to avoid inhibition of Hg2' antimicrobial activity. The 12 remaining beakers were left untreated as controls (control soil). The soils (control, sterile, treated) were fertilized with 30 mL of a nutrient solution bringing 0.8 mg N (g of soil)-' (NO3NH4), 1.2 mg of P (g of soil)-' (Na2HP04 and KHzPO~as phosphate buffer), and 0.4 mg of K (g of soil)-' (KH2P04), a fertilization similar to that used in liquid batch cultures ( 1 1 ) and well above the rates recommended in landfarming practices (12). Such high values were chosen to avoid any * Corresponding author Fax: 33-1-40-79-35-94. + Ecole Nationale Superieure d'ilgronomie et des Industries Alimentaires. Museum National d'Histoire Naturelle.
*
0013-936W95/0929-1615$09.00/0
D 1995 American Chemical Society
VOL. 29, NO. 6,1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
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TABLE 1
Composition of Experimental Agricultural Soil and Drilling Cuttings PH silt (%) clay (%) sand (%I organic matter (%) total N (q/o0) C/N P205 (%o) (Olsen) total Ca (%I fuel oil (%)
soil
drilling cuttings
5.1 52 29 19 2.62 1.2 8.4 0.156 0.3 0
9.1 20 10 70 12.4 0.6 103 0.19 10.5 5.7
2500 3000
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olfi 0
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I
60
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I
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.
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I
180
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I
240
I.'
I
300
T I M E (DAYS)
nutrient limitation in microbial activity. Also the amount of P was high enough to overcome immobilization of the nutrient by Ca2+ions of drilling cuttings. The microcosms were incubated for 270 days at 24 f 1"C. Periodic additions of sterile water maintained humidity at 30% of the waterholding capacity. To determine if the inorganic part of the drilling cuttings has an influence on biodegradation rates of HC, a similar series of beakers was prepared to which the same amount of the same fuel oil was added, without the mineral fraction. Chemical Analyses. At days 0,8,16,30,60,90,150,210, and 270, four beakers of treated soil and one of sterile and control soils were sampled to measure the residual concentration and composition of the hydrocarbons. The method, previously described (8,111,consisted in the drying of the soil samples for 12 hat 60 "C and the Soxhlet extraction of the entire sample with chloroform during 8 h. The extract was purified by percolating on a 60-100 mesh Florisil column, which retained the polar lipids of the soil, and was evaporated in a preweighed dish. The residue was weighed, analyzed by quantitative computerized capillary gas chromatography (GC), and further separated in saturated, aromatic, and polar fractions by successive elution with 60 mL each of hexane, benzene, and methanol on a 15 x 1.5 cm chromatographic column filled with 100-200 mesh activated silica gel. The initial fuel oil used in drilling cuttings contained 60% saturates, 30%aromatics, and 10% polar compounds. The polar fraction of this oil was composed of resins with almost no asphaltenes. Each fraction was weighed and the aromatic fraction was analyzed by GC . Before GC analysis, n - 1 eicosene was added as an internal standard in total oil and aromatic fraction to permit the quantification of all individual compounds separated by GC. GC conditions were as follows: the Delsi DI 200 chromatograph was equipped with a direct injection port and a FID detector both set at 350 "C; carrier gas was helium under 0.8 bar; column was a CP Si1 5 CB (Chrompack) capillary column (50 m x 0.32 mm, film thickness 0.25 pm): temperature programming was 100-320 "C, 3 "C/mn. Acquisition and further numerical treatment of data were performed using custom-made computer programs. GC-resolved aromatic compounds were identified on reference analyses by gas chromatography/mass spectrometry using the same chromatograph and a ITD 800 (Finnigan MAT) mass detector. Statistical Analysis. The decrease with time in total fuel oil and fractions was modeled using statistical curve 1616
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 6 , 1995
FIGURE 1. Hydrocarbon concentration in oil-treated sterile soils (O),oil-treated soils (01,and control soils (0).
fitting. The determination of best fit curves between linear, logarithmic, exponential, hyperbolic, or homographic adjustments was achieved considering the highest value of correlation coefficient r z. Microbiological Analyses. Numbers of total heterotrophic bacteria (THB) and hydrocarbon-utilizing bacteria (HUB)were determined on each soil sample with the most probable number method MPN using three tubes per dilution. For THB, the tubes of trypcase-soja (30 g L-l) medium were inoculatedwith decimal dilutions of soil and incubated at 24 f 1 "C for 3 days. For HUB, the mineral medium MM was composed of KH2P040.68 g L-' , Na,HP04 1.79 g L-I , MgS04 0.35 g L-I , N03NH4 1 g L-l, CaC12, FeS04 0.4 mg L-I, and 0.1 mL of a solution of 100 mg L-' of H3B04,MnS04,ZnS04, CuS04, and CoC12, After sterilization, 0.1 mL of sterile crude oil was added to each tube. Inoculation was done like for THB by decimal dilutions and incubation was run for 21 days at 24 f 1 "C . The number of viable microorganisms was obtained from standard MacCrady tables after examination of growth positive tubes. Strains of HUB and hydrocarbon-utilizing fungi HUF were isolated from treated and control soils during the experiment at day 8 and day 150. Serial dilutions of soil suspensions were plated on specific media supplemented with crude oil at the rate of 5 mL L-I and thoroughly shaken to form an emulsion. For HUB, the medium was composed of MM and agar 20 g L-l, for HUF, the medium was composed of KC1 250 mg L-I, NaH2P041 g L-I, MgS04 0.5 g L-l, N03NH41 g L-l, chloramphenicol 100 mg L-I, and agar 20 g L-l. Plates were incubated at 24 i 1 "C for 21 days. At the end of the incubation, each individual colony was cultured in 20 mL of liquid HUF or MM medium in 20 x 200 mm tubes to which 0.1 mL of crude oil was added. After another 21 days of incubation, the strains that showed a visible growth were scored as potentially able to use petroleum hydrocarbons as the carbon and energy source. Fungal strains were identified according to general principles of fungal classification. Bacteria were testedwithAP1 (Bio-Mkrieux)micromethods and identified on the basis of Bergey's Manual of Systematic Bacteriology (13, 14).
Results Hydrocarbons in Control and Sterile Soils. Control soils contained an initial concentration of biogenic HC around 50 pg g-I. No qualitative nor quantitative change was
TEMPERATURE FIGURE 2. Gas chromatographic analysis of biogenic hydrocarbons in control soils.
recorded during incubation (Figure 1). Identified compounds were principally odd n-alkanes in the nC29-nC33 range (Figure 2). The sum nC29 nC3 1 nC33 was in the range of 1.4 pg g-l. The carbon preference index CPI, i.e., the ratio C odd n-alkanes/E even n-alkanes was 7.6. In sterile soils, evaporation of the lightest (nC11-nC13) compounds during analytical processing of the samples reduced initial content from 2670 to 2240pg g-' (16%loss). This concentration then remained constant (Figure 11, thus indicating that no abiotic loss occurred during incubation. Therefore, any additional loss recorded in the treated soil was attributed strictly to biodegradation. Hydrocarbonsin Treated Soils. Results are given for microcosms treatedwith drilling cuttings only. The results for microcosms with fuel oil were nearly identical without any statistically significant difference and are not detailed here. Concentration of HC in treated soils decreased sharply during incubation (Figure 1). After the initial evaporation, HC (nC14-nC27) concentration was 2190 pg g-l. At day 270, it was reduced to 547pg g-l, corresponding to the biodegradation of 75% of total HC. The decrease in HC concentration was logarithmic with time (HCpg g-I = 1694-411 log t; r 2 = 0.977). Biodegradation of 50% occurred during the first two months. Drilling cuttings HC could be quantitated by GC (Figure 3). In the saturated fraction, all aliphatics (n- and branched alkanes) were separated by GC; they represented 40% of the fraction; 10%of the aromatic fraction was separated by GC. Nonseparated compounds were included in the unresolved complexmixture(UCM). The n-alkanes (nC14nC27) were rapidly and quite completely removed in 16 days (Figures 3 and 41, following a linear decrease with time (n-alkane pg g-l = 278-17.5 t; r 2 = 0.991). The degradation rate was 17.5 pg g-' n-alkanes per day. No difference in the rate of metabolization of odd and even n-alkanes was recorded. It is worth noting that biogenic n-alkanes nC29-nC33 were not degraded during the experiment (Figure 3). Like in the control soil, the sum nC29 + nC31 nC33 remained constant with a value of 1.4 & 0.1 pg g-l. After 270 days, the only remaining n-alkanes in the soil were biogenic, with small amounts of nC14-nC25, less than 0.5 pg g-l each. Branched alkanes were biodegraded more slowly than n-alkanes (Figures 3 and 4). Their degradation rate was two times lower than the n-alkanes (8.3 pg g-' branched
+
+
+
alkanes per day), and the decreasing curve was also linear with time (branched alkanepg g-' = 354-8.3 t ;r = 1.000). Isoprenoids pristane and phytane resisted until day 60 but were finally totally degraded. At the end of the incubation, they were almost completely metabolized, only 4.2% remained. UCM was less degraded than the aliphatics (Figure 3). During the first 60 days, 57% of the total UCM compounds were metabolized. During the following 90 days, 24% of the remaining compounds were also degraded. Little biodegradation occurred during the 150-270 day period. The left part of the UCM was more intensively degraded than the right part, indicating that lower molecular weight compounds were preferably used by microorganisms. As shown on Figure 5, the biodegradationofthe saturated fraction was logarithmic with time (saturated HC pg g-' = 1058-260 log t; r = 0.939). At day 270, 22% of the total amount remained in the soil. The aromatic fraction was mainly composed of polycyclic aromatic HC (PAH) beginning in the molecular weight range of trialkylnaphthalenes. The initial concentration of aromatics was 570 pg g-l; at the end of the experiment, 165pg g-' remained (29%of the initial weight). GC identifiable compounds were all biodegraded following different rates according to their chemical structure (Figure 3 and Table 2). The biodegradation of the aromatic fraction was maximal in the first two months and nearlynil duringthe late part of the experiment, following the function: aromatic HCpg g-' = 535-127 log t; r 2 = 0.72 (Figure 5). The resin fraction represented initially 10% of the total amount of oil. During the experiment, no significant change in the concentration of this fraction was observed (Figure 5). Microbial Changes. In control soils, temperature and fertilization increased THB and HUB respectively 4.4 and 2.1 times (Figure 6). No significant variations were recorded during incubation, the levels of the populations being almost constant in the range of 3 x lo8 cells for THB and 3 x lo6 cells for HUB. the addition of drilling cuttings HC produced strong changes in the numbers of viable microorganisms. In 16 days, a 1000-fold increase in HUB and a 100-fold increase in THB were observed in treated soils in comparison to controls. Eight days after HC incorporation, bacteria adapted to HC assimilation represented 56% of the total counts. HUB and THB decreased regularly after 16 VOL. 29, NO. 6,1995 /ENVIRONMENTAL SCIENCE &TECHNOLOGY
1617
a TO
UCM
D 30
T O T A L HYDROCARBONS
AROMAT I CS
FIGURE 3. Gas chromatographic analyses of drilling cuttings of hydrocarbons. The numbers represent the carbon numbers of ,alkanes. UCM, unresolved complex mixture; IS, internal standard ( n - 1 eicosane); NPH, naphthalenes; FLU, fluorenes; PHN, phenanthrenes; DBT, dibenrothiophenes; TO, initial; D30-0270. day of experiment.
days and 60 days of incubation. At day 270, both HUB and THB populations had returned to levels similar to controls. Identification of Hydrocarbon Degrading Strains. Numerous strains of hydrocarbon degraders (bacteria and fungi) were isolated and identified (Table 3). Most of the 1618
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 6, 1995
bacterial strains belonged to the genera Pseudomonas, Micrococcus,Xanthomonas,Acinetobacter, Flavobacterium, Agrobacterium, Rhodococcus, and Arthrobacter. Active fungal strains belonged to the genera Aspergillus, Penicillium, Acremonium, Trichoderma, Gongronella, and Fusarium.
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