Physicochemical Properties and Biodegradability of Crude Oil

Environ. Sci. Technol. 1997, 31, 45-51

Physicochemical Properties and Biodegradability of Crude Oil KEIJI SUGIURA,† MASAMI ISHIHARA, TOSHITSUGU SHIMAUCHI, AND SHIGEAKI HARAYAMA*

compounds; the high molecular weight aromatics, resins, and asphaltenes generally being considered to be recalcitrant to biodegradation (2, 4). While there is considerable information on the microbial utilization of chemically defined hydrocarbons, there have been very few studies concerned with the relationship between biodegradability and physicochemical properties of petroleum. This paper elucidates the chemical species of petroleum that are recalcitrant to biodegradation.

Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi City, Iwate 026, Japan

Experimental Section

The biodegradation of four different crude oil samples, namely, Arabian light, Dubai, Maya, and Shengli, by Acinetobacter sp. T4 and by a microbial consortium called SM8 was examined. SM8 exhibited higher activity than Acinetobacter for the biodegradation of all four crude oil samples. The degree of biodegradation of crude oil components differed according to the crude oil, the saturated fraction being more susceptible to biodegradation than the aromatic fraction in all the crude oil samples. The extent of biodegradation by Acinetobacter and SM8 was found to be in the order of Arabian light > Dubai g Maya ) Shengli; the crude oil samples with higher API gravity being more susceptible to biodegradation. Saturated compounds of smaller molecular weight were preferentially degraded by both cultures. Acinetobacter could not degrade polycyclic aromatic compounds in the crude oil samples such as (alkyl)naphthalenes, (alkyl)phenanthrenes, (alkyl)fluorenes, and (alkyl)dibenzothiophenes. However, this strain was capable of degrading more than 10% of the molecules in the aromatic fraction of Arabian light crude oil. An NMR analysis demonstrated that the alkyl side chain of some aromatic molecules was degraded by this organism. In contrast, SM8 degraded the polycyclic aromatic compounds in the crude oil samples, the extent of degradation being in the order of Maya > Shengli > Arabian light > Dubai.

Introduction Accidental spillage of petroleum often provokes serious damage to the natural environment, and the microbial degradation of spilled oil is one major route in the natural decontamination process (1, 2). Petroleum contains hundreds of individual compounds, and its components are generally grouped into four classes according to their differential solubility in organic solvents: the saturates (n- and branched-chain alkanes and cycloparaffins), the aromatics (mono-, di-, and polynuclear aromatic compounds containing alkyl side chains and/or fused cycloparaffin rings), the resins (aggregates with a multitude of building blocks such as pyridines, quinolines, carbazoles, thiophenes, sulfoxides, and amides), and the asphaltenes (aggregates of extended polyaromatics, naphthenic acids, sulfides, polyhydric phenols, fatty acids, and metalloporphyrins) (3). Microbial attack on petroleum has been shown to occur toward n- and branched alkanes or toward several aromatic * Corresponding author telephone: 81-193-26-6544; fax: 81-19326-6592; e-mail address: [email protected]. † Present address: Chemical Research Center, Steel Development & Production Division, Kawasaki Steel Co. Ltd., Chuoh-ku, Chiba 260, Japan.

S0013-936X(95)00961-8 CCC: $14.00

 1996 American Chemical Society

Bacteria. A microbial consortium called SM8 has been isolated from sediment in Shizugawa Bay, Japan (5). This population was subcultured every 2 weeks on an NSW crude oil medium (see later for a description of this medium) at a concentration of 4000 mg/L. An n-tetradecane-degrading bacterium, strain T4, was isolated from the Pacific Ocean by an enrichment culture with 4000 ppm n-tetradecane as the source of carbon and energy. This bacterium has been identified as the Acinetobacter genus by conventional taxonomic tests and by the sequencing of gyrB (6). Crude Oil. Four different crude oil samples (Arabian light, Dubai, Maya, and Shengli) were purchased from Japan Energy Co. Ltd. (Tokyo, Japan). To remove the readily biodegradable components, 100 g of each crude oil sample was poured into a 1-L beaker and heated at 100 °C for 48 h with the unevaporated part (heat-treated crude oil) then being used in this study. By this treatment, the weights of the Arabian light, Dubai, Maya, and Shengli samples were reduced by 24.4%, 20.8%, 20.6%, and 10.6%, respectively. Media. (i) BSM. A basal salt medium (BSM) consists of NH4NO3 (1 g/L), NaCl (30 g/L), MgSO4‚7H2O (0.5 g/L), KCl (0.3 g/L), K2HPO4 (1.5 g/L), ferric citrate (0.02 g/L), and CaCl2 (0.2 g/L). (ii) NSW. Seawater was collected from Kamaishi Bay at a depth of 15 m. To prepare the natural seawater-based medium (NSW), fresh seawater was filtered through a MF Millipore filter (pore size ) 0.45 µM, Millipore, Bedford, MA), and NH4NO3 (1 g/L), K2HPO4 (0.2 g/L), and ferric citrate (20 mg/L) were added. The pH of the medium was adjusted to 7.6 by HCl, and heat-treated crude oil was added at a concentration of 4000 or 5000 mg/L to afford the NSW crude oil medium. The medium was autoclaved before use. Cultivation of Bacteria on Crude Oil Samples. Precultures of SM8 were grown at 20 °C for 10 days in BSM supplemented with 5000 mg/L of heat-treated Arabian light crude oil, while those of Acinetobacter sp. T4 were grown at 20 °C for 7 days in BSM supplemented with 5000 mg/L of n-tetradecane. To determine the biodegradability of each crude oil, Erlenmeyer flasks (500 mL) containing 100 mL of BSM supplemented with 5000 mg/L of crude oil (Arabian light, Dubai, Maya, or Shengli) were inoculated with 1 mL of a preculture of either the SM8 consortium or Acinetobacter sp. T4 and incubated at 20 °C for 28 days while shaking at 100 strokes/min. As controls, flasks each containing 100 mL of a non-inoculated crude oil medium were incubated similarly. Analysis of Crude Oil Samples. Crude oil in the culture fluid was extracted with chloroform, as described previously (7). The chloroform extraction was repeated until the brown color of the fluid was cleared. The chloroform was evaporated at 50 °C with a nitrogen stream to constant weight, and the extract was analyzed by the following methods. (i) Gravimetric Measurement. The amount of crude oil was determined by measuring the weight of the sample, the oil-degrading activity being evaluated by the decrease in weight compared with that of a control sample. (ii) Column Chromatography. A glass column (20 mm in internal diameter and 300 mm in length) was filled with

VOL. 31, NO. 1, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Characteristics of Heat-Treated Crude Oil Samples Arabian light APIa

gravity, viscosity (cp) at 20 °C aromaticityb n-C17/pristanec content saturatesd aromaticsd otherse

33.4 64 0.08 3.64 0.50 0.38 0.02

Dubai

Shengli

Maya

31.1 24.2 22.0 100 4600 5300 0.10 0.08 0.14 2.14 1.31 2.57 0.47 0.37 0.06

0.50 0.27 0.08

0.33 0.39 0.19

a American Petroleum Institute gravity. b Defined as the ratio of aromatic carbons to total carbons as calculated by the equation: (A1 + A2)/(A1 + A2 + A3) (see Figure 1 for the definition of A1, etc. in the 13C-NMR spectrum). c Ratio of n-heptadecane to pristane in the saturated fraction. d (Weight of each fraction separated by column chromatography) ÷ (weight before fractionation). e Weight in % of hexane-insoluble materials.

a slurry of activated silica gel (C200; Wako Pure Chemicals, Tokyo, Japan) suspended in n-hexane. A crude oil sample dissolved in n-hexane was loaded at the top of this column, and the column was successively eluted with 300 mL of n-hexane and 300 mL of toluene. The fractions eluted with these solvents are called the saturates and aromatics, respectively (8). The solvent present in each of these fractions was evaporated, and the residual oil was determined by gravimetric measurement. The recovery of the saturated and aromatic fractions is shown in Table 1. (iii) GC/FID. Analysis of the crude oil by gas chromatography (GC) was conducted in a fused silica capillary column (DB-5, 30 m long, 0.25 mm in diameter; J&W Scientific, Folsom, CA) connected to a gas chromatograph (GC-14A, Shimadzu, Kyoto, Japan). The operating temperature of the flame ionization detector (FID) was 300 °C, and that of the injector was 280 °C. The column temperature was set at 40 °C for the first minute, increased to 105 °C at a rate of 8 °C/min, kept at 105 °C for 1 min, and increased again to 280 °C at a rate of 2.5 °C/min. The carrier gas was helium at a flow rate of 1 mL/min. n-Decane was added to chloroform extracts as an internal standard, and the quantities of alkanes in the extracts were determined from the peak areas corresponding to these compounds on the gas chromatogram. (iv) GC/MS. Gas chromatography/mass spectrometry (GC/MS) was performed with a QP-2000A instrument (Shimadzu) fitted with a fused silica capillary column (DB-5, 30 m long, 0.25 mm in diameter; J&W Scientific). The conditions for the GC analysis were the same as those already described except that naphthalene was used as an internal standard. The GC/MS instrument was operated in the “selected ion monitoring” mode. The targets for analysis were naphthalene derivatives carrying an alkyl side chain (C1-C4); fluorene or its derivative carrying an alkyl side chain (C1); and phenanthrene, dibenzothiophene, or their derivatives carrying an alkyl side chain (C1-C2). (v) FDMS. Field desorption mass spectrometry (FDMS) was performed with a JMS-AX505H mass spectrometer equipped with a silicon emitter (JEOL, Tokyo, Japan) under the following conditions: emitter current, 0-40 mA; rate, 8 mA/min; cathode voltage, -7.0 kV; scan range, 0-2000 m/z; cycle time, 9 s/scan. (vi) 13C-NMR and 1H-NMR. 13C-NMR and 1H-NMR spectra were recorded by a Unity 300 spectrometer (Varian, Palo Alto, CA) at 300 MHz, the intensity of each chemical shift being determined relative to internal tetramethylsilane in CDCl3 (Wako Pure Chemicals). The aromaticity is defined as the ratio of aromatic carbon to total carbon. The amount of aromatic carbon was obtained from the integrated intensity of these peaks between 100 and 170 ppm on the 13C-NMR spectrum, while that of aliphatic carbon was found from the integrated intensity of peaks between 8 and 58 ppm (Figure

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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 1, 1997

1). The aromaticity index (fa) was calculated according to the equation: fa ) (amount of aromatic carbon) ÷ (amount aromatic carbon + amount of aliphatic carbon). Hydrogens on the alkyl side chains of an aromatic compound could be resolved into three classes by using 1H-NMR spectroscopy: the resonance positions of hydrogens bound to the R carbon of an alkyl side chain are between 2.0 and 4.2 ppm, those of hydrogens bound to the β or more-distant carbon are between 1.0 and 2.0 ppm, while those of hydrogens on terminal methyl groups are between 0.4 and 1.0 ppm (9; Figure 1). The average number of carbon atoms per alkyl substituent, n, was then calculated from the equation n ) (HR + Hβ + Hγ ) ÷ HR, where HR, Hβ, and Hγ are the amounts of hydrogen attached to the R carbons, to the β or more-distant carbons, and to the terminal methyl groups of the alkyl side chains, respectively (9). Determination of Viscosity of Crude Oil Samples. The viscosity of each crude oil sample was determined by a rotary viscometer (type E; Tokimec, Tokyo, Japan).

Results Characteristics of Crude Oil Samples. Many studies have established that low molecular weight alkane and aromatic compounds are readily biodegraded (3, 4). To remove these compounds from the crude oil samples, each was heated at 100 °C for 48 h. Characteristics of the heat-treated crude oil samples are shown in Table 1. The viscosity of the heattreated Maya and Shengli samples was much higher than that of the heat-treated Dubai and Arabian light samples. The aromaticity measured by 13C-NMR was higher in the Maya sample (14% aromatic carbons) than in the other samples (8-10% aromatic carbons). This result is in agreement with that from the column chromatographic analysis, in which the content of the saturated fraction in the Maya sample (33%) was lower than that of other samples (47-50%, Table 1). The ratio of linear C17 alkane to branched pristane was highest in the Arabian light sample, lowest in the Shengli sample, and intermediate in the other two samples. The heattreated crude oil samples were used in all the experiments subsequently described. Degradation of Crude Oil by the SM8 Consortium and Acinetobacter sp. T4. Time-course experiments demonstrated that the maximum extent of biodegradation of these crude oil samples in the cultures of the SM8 consortium and Acinetobacter sp. T4 was attained in about 10 days (data not shown). The degree of biodegradation of the crude oil samples was thus determined in 28-day cultures (Table 2). The SM8 consortium degraded between 19% and 34% of the whole crude oil samples, while Acinetobacter sp. T4 degraded a lesser amount ranging between 12% and 20%. The extent of biodegradation of the crude oil samples by both Acinetobacter sp. T4 and the SM8 consortium was found to be in the order of Arabian light > Dubai g Shengli > Maya (Table 2, Section A). The biodegraded crude oil samples were fractionated by column chromatography, and the degree of biodegradation of the saturated and aromatic fractions was each determined. As shown in Table 2, Section B, Acinetobacter sp. T4 degraded the saturated fraction of the samples ranging between 23% and 41%. This organism also degraded about 10% of the aromatic fraction of the Arabian light, Dubai, and Maya samples, but did not significantly degrade the aromatic fraction of the Shengli sample. The SM8 consortium degraded these crude oil samples to a greater extent: between 40% and 53% of the saturated fraction and between 11% and 18% of the aromatic fraction (Table 2, Section B). The GC/FID analysis showed that the n-alkanes (C14-C30) in the Dubai and Arabian light samples were completely degraded by the SM8 consortium and by Acinetobacter sp. T4. Although these n-alkanes in the Maya sample were completely degraded by the SM8 consortium, they were only

FIGURE 1. 1H- and 13C-NMR spectra of the aromatic fraction of the heat-treated Arabian light crude oil sample. (A) 1H-NMR spectrum. The ranges of Hr, Hβ, Hγ, and Ha are indicated below the ppm scale, where Hr, Hβ, Hγ, and Ha are respectively the fractions of r-alkyl hydrogens (2.0-4.2 ppm), alkyl hydrogens except the r-alkyl and terminal methyl hydrogens (1.0-2.0 ppm), terminal methyl hydrogens (0.4-1.0 ppm), and aromatic hydrogens (6.2-9.6 ppm). (B) 13C-NMR spectrum. The ranges of A1, A2 and A3 are indicated below the ppm scale, where A1, A2, and A3 are respectively the fractions of non-protonated aromatic carbons (130-170 ppm), protonated aromatic carbons (100-130 ppm), and the fraction of aliphatic carbons (8-58 ppm). degraded at the level of 70% by Acinetobacter sp. T4. The degree of degradation of the n-alkanes in the Shengli sample by both SM8 consortium and Acinetobacter sp. T4 was significantly reduced with the increasing number of the carbon chain. The branched alkanes (phytane and pristane) were almost completely degraded by the SM8 consortium,

while they were only partly degraded by Acinetobacter sp. T4 (Figure 2). The results of the GC/MS analysis demonstrated that naphthalene, fluorene, phenanthrene, dibenzothiophene, and their derivatives were degraded by the SM8 culture (Table 3). The biodegradability of these compounds was higher in the


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TABLE 2. Percentage Biodegradation of Four Crude Oil Samples Section A: Total Biodegradationa Arabian light Dubai

Acinetobacter sp. T4 SM8

20 ( 7 34 ( 7

14 ( 8 27 ( 4

Shengli

Maya

14 ( 2 25 ( 2

12 ( 10 19 ( 2

Section B: Biodegradation of Saturated and Aromatic Fractionsb Arabian light Dubai Shengli

Acinetobacter sp. T4 SM8

Maya

saturates

aromatics

saturates

aromatics

saturates

aromatics

saturates

aromatics

30 ( 5 49 ( 4

12 ( 4 16 ( 10

41 ( 4 53 ( 2

10 ( 1 11 ( 14

25 ( 1 40 ( 1

-1 ( 1 12 ( 3

23 ( 13 50 ( 12

7(1 18 ( 2

a The extent of biodegradation of the whole crude oil sample was determined by calculating: 100 × [1 - (weight of extracted oil from biodegraded culture) ÷ (weight of extracted oil from non-inoculated culture)]. The cultivation was carried out at 20 °C for 28 days. Data were obtained from four independent experiments. b The extent of biodegradation of the saturated or aromatic fraction was determined by calculating: 100 × [1 (weight of saturated or aromatic fraction separated by silica gel chromatography from extract of culture) ÷ (weight of saturated or aromatic fraction from extract of non-inoculated medium)]. Data were obtained from two independent experiments.

examined by using the FDMS method. Under the analytical conditions used (see Experimental Section), the fragmentation of the analyzed molecules was expected to be a minimum, the intensity vs m/z plots shown in Figure 3 most likely representing the molecular mass distribution of compounds in the Arabian light samples. In panels A and B, the molecular mass distribution of biodegraded species in the saturated fraction are compared with that in the undegraded sample. It is clear that saturated compounds whose m/z values are larger than 500 were not significantly degraded by either Acinetobacter sp. T4 or SM8 consortium. In panels C and D, the molecular mass distribution of aromatic compounds in the original sample and that of biodegraded molecules are presented. Unlike the biodegradation of the saturated compounds, aromatic molecules larger than 500 were also degraded by the SM8 culture (panel D).

FIGURE 2. Percentage biodegradation of the n-alkanes and branched alkanes. The percentage biodegradation of each compound was calculated by the equation: (peak area of the compound on the gas chromatogram of a biodegraded sample)/(peak area of the compound on the gas chromatogram of a control sample) × 100. Dubai (0), Arabian light (O), Maya (4), and Shengli (open cross). C14-C30, n-alkanes; PR, pristane; PHY, phytane. (A) Biodegradation by Acinetobacter sp. T4. (B) Biodegradation by the SM8 consortium. Maya and Shengli samples than in the Dubai and Arabian light samples. The biodegradability of the aromatic compounds was lower with increasing size of their alkyl side chain (Table 3). In contrast to the SM8 culture, the Acinetobacter sp. T4 culture did not significantly degrade these aromatic compounds (Table 3). Detailed Analysis of Biodegraded Arabian Light Sample. The Arabian light samples before and after biodegradation by either Acinetobacter sp. T4 or SM8 consortium were

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The molecular formula of an alkane can be expressed as CnH2n+2, and those of mono- and dicycloalkanes can be expressed as CnH2n and CnH2n-2, respectively. Since the saturated fraction of crude oil is known to contain no unsaturated hydrocarbons (10), the ratio of the amount of CnH2n or CnH2n-2 molecular species to the amount of CnH2n+2 species represents the relative amount of mono- or dicycloalkanes to the amount of alkanes. As shown in Figure 4, the ratio of CnH2n to CnH2n+2 in the non-degraded Arabian light sample was about 0.3 when n ) 21 and 22 and increased to about 0.8 when n ) 27 or more. Similarly, the ratio of CnH2n-2 to CnH2n+2 was lower than 0.1 when n ) 21 and increased to approximately 0.4 when n ) 27 or more. Both the ratio of CnH2n to CnH2n+2 and that of CnH2n-2 to CnH2n+2 were significantly higher in the biodegraded Arabian light sample, indicating that alkanes were more susceptible to biodegradation than the mono- and dicycloalkanes. Similarly, the ratio of CnH2n-2 to CnH2n+2 shows that monocycloalkanes were more susceptible to biodegradation than dicycloalkanes. The combination of 13C- and 1H-NMR spectroscopy has proved useful for analyzing the composition of various molecular species of petroleum (9, 11). We thus used this technique to analyze the biodegradation of the Arabian light sample. The aromaticity of the aromatic fraction was 0.38 before biodegradation and was increased to 0.43 and 0.41 after biodegradation by Acinetobacter sp. T4 and SM8 consortium, respectively (Table 4). This increase in the proportion of aromatic carbons suggests that the alkyl side chains of the aromatic molecules were preferentially degraded by these organisms. In agreement with this observation, the average number of carbon atoms per alkyl substituent was reduced from 4.22 to 3.88 and to 4.04 after biodegradation by Acinetobacter sp. T4 and SM8 consortium, respectively, again indicating that a diminution of the average length of lateral chains resulted from biodegradation.

FIGURE 3. Molecular mass distribution of compounds in the saturated and aromatic fractions from the Arabian light crude oil sample. Crude oil was extracted from an Acinetobacter sp. T4 culture, an SM8 culture, and a control sample and separated into the saturated and aromatic fractions. The weights of the saturated and aromatic fractions from the Acinetobacter sp. T4 culture were 70% and 88%, respectively, of the control sample, while those from the SM8 culture were 51% and 84%, respectively, of the control sample. These samples were analyzed by FDMS, and the integrated signal intensity (in %) within each m/z 10 band (e.g., m/z 200-210, m/z 210-220, etc.) was calculated. The signal intensities in % of the biodegraded samples were corrected for the reduced weight, e.g., the signal intensities in % of the saturated fraction from the Acinetobacter sp. T4 culture were multiplied by 0.70. The difference between the signal intensities of the control sample (O) and the corrected signal intensities of a biodegraded sample calculated by the equation (% intensity in the control sample) - (% intensity in a biodegraded sample) × (weight of the biodegraded sample relative to the control sample) represents the signal intensities of biodegraded molecules (b). (A) Intensities of compounds in the non-degraded saturated fraction (O) and those of saturated compounds degraded by Acinetobacter sp. T4 (b). (B) Intensities of compounds in the non-degraded saturated fraction (O) and those of saturated compounds degraded by the SM8 population (b). (C) Intensities of compounds in the non-degraded aromatic fraction (O) and those of aromatic compounds degraded by Acinetobacter sp. T4 (b). (D) Intensities of compounds in the non-degraded aromatic fraction (O) and those of aromatic compounds degraded by the SM8 population (b).

Discussion Few studies have compared the biodegradability of different petroleum samples. Westlake et al. (12) have investigated the biodegradation of four different Arctic crude oil samples at two different temperatures of 4 and 30 °C and demonstrated that the chemical composition of crude oil and the cultivation temperature had a marked effect on the composition of the bacterial consortium and its efficacy in biodegradation. The biodegradability of two refined products has been compared with that of two crude oil samples (13), and similar conclusions to those in the above-mentioned study were drawn: the microbial species found in cultures grown on different oil samples were different, and South Louisiana crude oil, which is a low-sulfur and high-saturate type, was found to be more susceptible to biodegradation than the Kuwait crude oil. More recently, McMillen et al. (14) observed that biodegradability of crude oil was proportional to the square of American Petroleum Institute (API) gravity. In our results, the correlation between the biodegradability (Table 2, Section A) and the square of API (Table 1) (r ) 0.82 for Acinetobacter sp. T4 and r ) 0.92 for the SM8 consortium) as well as that between the biodegradability and the viscosity (Table 1) (r ) 0.69 for Acinetobacter sp. T4 and r ) 0.83 for the SM8 consortium) were observed.

GC-resolved n-alkanes (C14-C30) in the Arabian light and Dubai samples were completely degraded, but some of these compounds in the Maya and Shengli samples were only partly degraded by Acinetobacter sp. T4. The degree of biodegradation by Acinetobacter sp. T4 of pristane and phytane in different crude oil samples also varied, ranging between 15% and 60% of the initial concentration. In other words, the same compounds in different crude oil samples were degraded to different degrees by the same organism. Thus, the biodegradability was not solely determined by the chemical structure but by other factors as well. The most simple interpretation of the observation is that the bioavailability of these alkanes in different crude oil samples was different. For example, a fraction of these alkanes in the Maya and Shengli samples may be embedded in aggregated matrixes and escaped the biodegradation process. Cycloalkanes make up a large proportion of certain types of crude oil, having a structure involving one or several rings of cyclopentane or cyclohexane to which alkyl side chains are attached. In this study, the biodegradability of cycloalkanes was found to be lower than that of linear alkanes (Figure 4). It is known that cyclopentane and cyclohexane are hardly biodegradable by pure cultures (15), so the presence of the


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TABLE 3. Percentage Biodegradation of Some Aromatic Compounds in Four Crude Oil Samples

A

intensity crude oil

biodegradation (%)b

moleculea

abiotic control

standard C1-Np C2-Np C3-Np C4-Np Fl C1-Fl DBT C1-DBT C2-DBT Phe C1-Phe C2-Phe

52855 0 10901 39050 35252 1021 4997 20027 64992 96163 7423 16698 21939

standard C1-Np C2-Np C3-Np C4-Np Fl C1-Fl DBT C1-DBT C2-DBT Phe C1-Phe C2-phe

39084 866 19158 44670 27613 1948 5837 16246 30880 42143 8350 15355 22380

Dubai 38136 757 17541 44185 24433 1821 4783 15792 28520 41335 8011 16739 19363


42926 800 8018 14546 10795 933 2644 1200 3037 3829 6527 9337 10662

Shengli 56755 1315 12130 19715 14384 1228 3516 1639 4625 5073 9159 14219 16086


51476 1338 18234 28572 16851 1377 5368 13381 35320 48310 7646 18201 18585

T4

SM8

T4

SM8

NDc -18 -6 6 -19 5 4 12 14 2 21 17

ND 93 68 49 82 39 88 37 20 99 54 18

43360 0 8399 42295 28096 806 5552 4371 30944 52052 452 17067 23798

10 6 -1 9 4 16 0 5 -1 2 -12 11

100 60 15 8 63 14 76 10 -11 95 0 4

49838 0 0 149 8385 90 141 0 370 3047 0 0 8601

-24 -14 -3 -1 0 -1 -3 -15 0 -6 -15 -14

100 100 99 33 92 95 100 90 31 100 100 31

Maya 44785 47982 1365 0 17074 0 26780 1332 15807 9994 1343 0 4988 399 11921 140 29955 2477 38623 24913 6872 169 13408 951 14167 9511

-17 -8 -8 -8 -12 -7 -2 3 8 -3 15 12

100 100 95 36 100 92 99 92 45 98 94 45

Arabian Light 38682 49222 0 0 9409 686 30171 11586 24378 16671 887 169 3474 2838 14112 2203 41758 38367 60876 71355 5337 43 9652 7100 13362 16753

a Np, Fl, DBT, and Phn represent naphthalene, fluorene, dibenzothiophene, and phenanthrene, respectively. C1-C4 represent carbon numbers of the alkyl groups in alkylated polycyclic aromatic hydrocarbons. Compounds C1-Np, C2-Np, C3-Np, C4-Np, Fl, C1-Fl, DBT, C1DBT, C2-DBT, Phn, C1-Phn, and C2-Phn were characterized by the signals at m/z 142, 156, 170, 184, 166, 180, 184, 198, 212, 178, 192, and 206, respectively. Naphthalene (m/z 128) was used as a standard. b The percentage biodegradation was calculated by the equation 100 - 100 × (A - B)/A where A ) (signal intensity of an aromatic molecule in a control sample)/(signal intensity of the standard in a control sample) and B ) (signal intensity of the aromatic molecule in a biodegraded sample)/(signal intensity of the standard in a biodegraded sample). c Not detected.

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B

C

FIGURE 4. Changes in the molar ratio of alkanes (CnH2n+2), monocycloalkanes (CnH2n), and dicycloalkanes (CnH2n-2) before and after biodegradation. Open boxes, the SM8 culture; hatched boxes, the Acinetobacter sp. T4 culture; filled boxes, control (without bacterial inoculation). cycloalkane rings may affect the biodegradation of their alkyl side chains. The FDMS analysis demonstrates that saturated compounds of smaller molecular weight were preferentially degraded by Acinetobacter sp. T4 and SM8 consortium (Figure 3). An important fraction of the biodegraded molecules may correspond to n-alkanes (C14-C30), which were shown by the GC/FID analysis to be efficiently degraded by these organisms (Figure 2). Saturated compounds having a molecular weight larger than 500 were not degraded by these organisms (Figure 3), this size corresponding to the exclusion size for passage through the outer membrane of Gram-negative bacteria (16). Acinetobacter sp. T4 and the major constituents of the SM8 consortium are Gram-negative (5); thus, the outer membrane permeability may be one of the major factors to determine biodegradability. It is shown in Figure 4 that the proportion of cycloalkanes to linear alkanes in the saturated fraction gradually increases as the number of carbon atoms increases. Thus, the percentage of recalcitrant molecules increases as the size of the molecular increases. This may be one of the reasons why the higher molecular weight compounds in the

TABLE 4. Average Molecular Structure in Aromatic Fraction of Arabian Light Crude Oil Sample before and after Biodegradation 1H-NMRa

control Acinetobacter SM8

13C-NMRa

Ha

Hr

Hβ

Hγ

A1

A2

A3

fab

nc

0.123 0.127 0.128

0.208 0.225 0.216

0.495 0.489 0.489

0.174 0.160 0.168

0.153 0.100 0.076

0.227 0.328 0.329

0.620 0.572 0.595

0.38 0.43 0.41

4.22 3.88 4.04

a The amount of the hydrogen or carbon atom in each fraction was determined by NMR as shown in Figure 1. b The aromaticity, fa, is defined as the ratio of aromatic carbon to total carbon, and was calculated by the equation: fa ) (A1 + A2)/(A1 + A2 + A3). c The average number of carbon atoms per substituent, n, was calculated by the equation n ) (HR + Hβ + Hγ)/HR (9).

saturated fraction were less susceptible to biodegradation. Solubility may be another factor to influence biodegradability, it being known that the solubility, and hence the accessibility to catabolic enzymes, of a hydrocarbon molecule decreases as the number of its carbon atoms increases. The extent of biodegradation of the aromatic fraction by Acinetobacter sp. T4 ranged between nil and 12%, depending upon the crude oil sample. This strain did not express any enzyme activity for the degradation of benzene, toluene, naphthalene, or phenanthrene (our unpublished results). In fact, naphthalene, fluorene, dibenzothiophene, phenanthrene, and their derivatives in the crude oil samples were not significantly degraded by this strain (Table 3). Therefore, Acinetobacter sp. T4 may degrade the alkyl side chains but not the aromatic rings of compounds in the aromatic fraction. This inference is confirmed by the results of 13C- and 1HNMR spectroscopy: the aromaticity of the aromatic fraction of the Arabian light sample was increased, while the average number of carbon atoms per alkyl substituent was decreased by biodegradation (Table 4). In contrast to Acinetobacter sp. T4, the SM8 consortium degraded naphthalene, phenanthrene, fluorene, dibenzothiophene, and their structural analogues. The degree of biodegradation of these molecules varied according to the crude oil types, being in the order of Maya > Shengli > Arabian light > Dubai. It is interesting to observe that the biodegradability of these aromatic molecules in the Maya sample was the highest, while that of the saturated fraction in the same crude oil samples was the lowest. It is conceivable that, in the biodegradation of crude oil samples containing a higher proportion of easily assimilable alkanes, the proportion of alkane-degrading bacteria increases in the SM8 microbial population and, consequently, the growth and activity of the aromatic-degrading bacteria decrease. The results of the FDMS analysis show that the molecular mass was not a critical factor for the biodegradation of aromatic molecules by the SM8 culture (Figure 3). We found that Sphingomonas bacteria in the SM8 consortium were mainly responsible for the biodegradation of the aromatic molecules (our unpublished data). This group of bacteria possessing sphingolipids in their outer membrane structure can adhere to the surface of an insoluble substrate and degrade it (17). It is thus probable that Sphingomonas can degrade hydrocarbons of high molecular weight and low solubility, and this possibility is currently being investigated in our laboratory.

Acknowledgments We thank Masafumi Goto and Masayoshi Asaumi for their many useful suggestions and Kaori Sasaki and Etsuro Sasaki for their technical assistance. This work was supported by a grant from New Energy and Industrial Technology Development Organization.

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)

(15)

(16)

(17)

Prince, R. C. Crit. Rev. Microbiol. 1993, 19, 217. Zobell, C. E. Bacteriol. Rev. 1946, 10, 1. Leahy, J. G.; Colwell, R. R. Microbiol. Rev. 1990, 54, 305. Atlas, R. M. Microbiol. Rev. 1981, 45, 180. Venkateswaran, K.; Iwabuchi, T.; Matsui, Y.; Toki, H.; Hamada, E.; Tanaka, H. FEMS Microbiol. Ecol. 1991, 86, 113. Yamamoto, S.; Harayama, S. Int. J. Syst. Microbiol. 1996, 46, 506. Goto, M.; Kato, M.; Asaumi, M.; Shirai, K.; Venkateswaran, K. J. Mar. Biotechnol. 1994, 2, 45. JPI (Japan Petroleum Institute) Standard (in Japanese); Japan Petroleum Institute: Tokyo, 1983. Clutter, D. R.; Petrakis, L.; Stenger, R. L.; Jensen. R. K. Anal. Chem. 1972, 44, 1395. Leffler, W. L. Petroleum Refining for the Non-Technical person; PennWell Books: Tulsa, OK, 1985. Ali, M.; Bukhari, F. A.; Hasan. M. Fuel Sci. Technol. Int. 1989, 7, 1179. Westlake, D. W. S.; Jobson, A.; Phillippe, R.; Cook, F. D. Can. J. Microbiol. 1974, 20, 915. Walker, J. D.; Petrakis, L.; Colwell, R. R. Can. J. Microbiol. 1976, 22, 598. McMillen, S. J.; Requejo, A. G.; Young, G. N.; Davis, P. S.; Cook, P. D.; Kerr, J. M.; Gray, N. R. In Microbial Processes for Bioremediation; Hinchee, R. R., Brockman, F. J., Vogel, C. M., Eds.; Battelle Press: Columbus, OH, 1995; p 91. Morgan, P.; Watkinson, R. J. In Biochemistry of Microbial Degradation; Ratledge, C., Ed.; Kluwer Academic Publishers: London, England, 1994; p 1. Nikaido, H.; Vaara, M. In Escherichia coli and Salmonella typhimurium; Neidhardt, F. C., Ed.; American Society for Microbiology: Washington, DC, 1987; Vol. 1, p 7. Stefan, S.; Rolf-Michael, W.; Dirk E.; Heinz, W.; Wittko, F. Appl. Environ. Microbiol. 1992, 58, 2744.

Received for review December 29, 1995. Revised manuscript received May 23, 1996. Accepted August 1, 1996.X ES950961R X

Abstract published in Advance ACS Abstracts, November 1, 1996.


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Physicochemical Properties and Biodegradability of Crude Oil

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