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Variations of Acidic Compounds in Crude Oil during Simulated Aerobic Biodegradation: Monitored by Semi-Quantitative Negative-Ion ESI FT-ICR MS Yinhua Pan, Yuhong Liao, and Quan Shi Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b02167 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 4, 2017
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Variations of Acidic Compounds in Crude Oil during Simulated Aerobic Biodegradation: Monitored by Semi-Quantitative Negative-Ion ESI FT-ICR MS
Yinhua Pan,†,‡,§ Yuhong Liao,*,† Quan Shi,‖
†
The State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, 511 Kehua Street, Wushan Road, Tianhe District, Guangzhou, GD 510640, China
‡ §
University of Chinese Academy of Sciences, Yuquan Road, Beijing 100049, China Sinopec Key Laboratory of Petroleum Accumulation Mechanisms, Wuxi Research Institute of Petroleum Geology, Sinopec Petroleum Exploration & Production Research Institute, 2060 Lihu Road, Binhu District, Wuxi, Jiangsu 214000, China
‖
State Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing 102249, China
* Corresponding Author. Telephone: +86-20-8529-1567. Fax: +86-20-8529-0706. Email:
[email protected].
ABSTRACT: Simulations of aerobic biodegradation have been widely employed to investigate the mechanisms of crude oil biodegradation in geological environments. In this study, a simulated biodegradation experiment was performed with crude oil under aerobic conditions, in which n-alkanes were nearly depleted, thus providing an opportunity to study the biodegradation mechanisms of n-alkanes in crude oils. The sequences of biodegraded oils with a slight to moderate degree of biodegradation were characterized by negative-ion electrospray (ESI) Fourier transform ion cyclotron resonance mass
spectrometry (FT-ICR
MS) and
gas
chromatography (GC).
Semi-quantitative results on the molecular compositions of heteroatom classes were obtained by co-injection of internal standards. The biodegradation mechanisms for 1
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n-alkanes and n-fatty acids, as well as some other heteroatomic compounds are discussed. Evidence from FT-ICR MS and GC analyses of biodegraded oils indicates that n-alkanes can be progressively biodegraded to n-fatty acids through β-oxidation, or to hydroxycarboxylic and dicarboxylic acids though ω-oxidation. The O3 class species which have a high relative abundance in the carbon number range of C33–C38 with Double Bond Equivalent (DBE) of 1–3 were assigned and speculated to be bacterial metabolites, which could be a conspicuous indicator of bacterial activity. Neutral nitrogen compounds, such as carbazoles, exhibited a very slight decrease in the stage of biodegradation that was investigated.
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1. INTRODUCTION Biodegradation, as a ubiquitous secondary alteration in reservoirs, is a principal process that forms most of the heavy crude oils.1,2 Sequential and systematic variations in the chemical compositions of crude oils, such as the preferential removal of hydrocarbons and the relative preservation of non-hydrocarbons, are commonly observed in in-reservoir biodegradations,3–7 as well as in laboratory simulations.8–12 These changes give rise to an increase in oil density, acidity, and viscosity, which produces negative economic consequences. In general, studies on the biodegradation of crude oil, both on the negative and positive sides, are of concern in petroleum production and oil refining, as well as with studies in other fields such as organic geochemistry, geobiology and environmental science. In comparison with conventional crude oils, biodegraded oils are richer in heteroatomic (i.e., nitrogen, sulphur and oxygen) compounds, which may cause many problems such as product instability during its transportation and storage, catalyst poisoning, environmental pollution and corrosion of some equipment used in oil refining.13–16 The influence of biodegradation on the composition and physical properties of petroleum hydrocarbons are well documented.17–19 It has been proposed that the general order of bio-resistance of various biomarker compound classes mostly follows the sequence: n-alkanes (least resistant) < acyclic isoprenoids < steranes < hopanes < diasteranes < aromatic steroids (most resistant).20–25 Based on previous work, Peters and Moldowan25 summarized and proposed a qualitative indicator of oil biodegradation (i.e., the Peters and Moldowan scale) according to a sequence of the removal of saturated hydrocarbons. Generally, anaerobic biodegradation of crude oils is a common occurrence in subsurface oil reservoirs.1,26 The in-reservoir biodegradation is a very slow process. Many millions of years may be needed for the removal of petroleum hydrocarbons. The degradation rate of crude oils is essentially determined by the reservoir temperature, nutrient supply, water salinity and the oil-degradation level.2,27 Aerobic biodegradation, 3
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however, is much faster than anaerobic biodegradation due to a sufficient amount of electron acceptors and appropriate nutrients, thus it is widely applied to study the biodegradation of crude oil. However, biodegradation of crude oil under aerobic conditions to some extent might differ from that under anaerobic conditions in a biodegradation pathway regarding the availability of microorganism types/species and metabolites. For example, it has been proposed that the aerobic biodegradation of n-alkanes mainly involves terminal and subterminal oxidation, with the predominant biodegradation products being carboxylic acids and alcohols, in which the alcohols are subsequently oxidized to acids.28–31 In a further degradation process, carbon chains of fatty acids can be shortened by two carbon atoms compared to the original n-alkanes through β-oxidation.32–36 With respect to anaerobic biodegradation, n-alkanes are mainly activated at the subterminal carbon of the alkane by the addition of fumarate, yielding a substituted succinate, and also can be further degraded by β-oxidation.37–39 In addition, metabolites that are indicative of the anaerobic degradation of hydrocarbons from degraded oils have been reported in the literature,26 improving the cognition of the biodegradation of crude oil. However, in vitro experiments that were performed on formation water and oils by da-Cruz et al.40 indicated that the biodegradation of petroleum was more likely to be a joint achievement of both aerobic and anaerobic bacterial consortia. Accordingly, the aerobic biodegradation of crude oil has the potential to portray and elucidate the probable processes and mechanisms of hydrocarbon biodegradation in reservoirs. Gas chromatography (GC) has demonstrated excellent sensitivity and has been applied extensively in the analysis of the acidic compounds in crude oils. As an example, methyl esterification is typically employed to characterize fatty acids in which their derivative products are amenable to GC analysis.41,42 However, sample pre-treatment for multi-step fractionation and derivatization is time-consuming and very costly. In addition, not all of the acids in oils can be detected because of the limitations in recovery rates, 4
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derivative efficiency and chromatographic and mass spectrometric resolution. Electrospray (ESI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has provided an established tool for the study of heteroatomic species in crude oils without prior chromatographic fractionation.43–45 ESI FT-ICR MS has been successfully applied to characterize the distribution of polar compounds in biodegraded oils and to study their biodegradation mechanisms.7,46,47 In this work, by employing GC and ESI FT-ICR MS analyses, we report the variations in the distribution of acidic and neutral polar compounds, along with saturated hydrocarbons
during
aerobic
biodegradation
simulations.
The
biodegradation
mechanisms of n-alkanes and n-fatty acids, as well as some other heteroatomic compounds, were explored. 2. EXPERIMENTAL SECTION 2.1. Aerobic Biodegradation Simulation Aerobic biodegradation was studied by diffusing oil onto an aqueous phase surface using mineral salts medium (MSM) as a nutrient substance. The crude oil was derived from the Zhaogu-1 oil well (3164–3191 m) which is located at the central uplift of the Liaohe Basin, China. The crude oil had a high wax content, and its saturated oil fraction accounted for up to 78.5 wt %. The bacterial consortium was enriched from a petroleum contaminated soil that was obtained from the Jalaid Banner from Innner Mongolia and then selectively screened three times in a MSM containing crude oil as the sole source of carbon. By using repeated plate streaking and subsequent standard morphological and biochemical taxonomic tests that are referred to the literature,48 the isolated strains from the soil samples were preliminarily identified as Pseudomonas, Bacillus and Burkholderia. The MSM contained (per liter) 1.0 g of K2HPO4·3H2O, 1.0 g of KH2PO4, 0.5 g of MgSO4·7H2O, 1.0 g of NH4NO3, 0.02 g of CaCl2 and a trace amount of FeCl3. The pH was adjusted to 7.0 before autoclaving. The enrichment medium contained (per liter) 10 g of peptone, 10 g of beef extract, 5 g of NaCl; and the pH of the solution was 5
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adjusted to 7.0. Each inoculum contained 1.0 g of crude oil and 2 mL of bacteria and was added to 100 mL of medium in a flask. The flasks were incubated at 30 °C and shaken at 180 rpm on a rotary shaker. A corresponding blank control group without a bacterial inoculum was also conducted. The degradation was terminated after 1, 2, 4, 6 and 8 weeks. The biodegraded oil/water sample was transferred to a separatory funnel that contained 10 mL of dichloromethane (DCM) to sterilize the bacteria and extract the organic phase. The extraction was repeated five times until the DCM extract was colorless. All of the extracts were combined and the DCM was removed by rotary evaporation to obtain the biodegraded oil. The extent of oil biodegradation was determined gravimetrically and was calculated as the percentage weight loss of oil after a background subtraction of the blank control group. All chemicals used in this study were of analytical grade. Table 1 lists some parameters of the biodegraded oils. As the biodegradation time proceeds, the degradation extent increases gradually while the pH of the aqueous phase decreases to approximately 6.1. 2.2. SARA Fractionation of Crude Oils The biodegraded oil was separated into maltene and asphaltene fractions using a deasphaltening procedure that is described in detail by Liao et al.49 The maltene fraction was fractionated into saturated, aromatic and resin fractions by silica gel/alumina column chromatography and eluted with n-hexane, DCM/n-hexane (3:1, v/v) and DCM/methanol (2:1, v/v). The saturated fraction was analyzed by gas chromatography-mass spectrometry (GC-MS) and GC. 2.3. GC-MS and GC Analyses The GC-MS analysis was performed using a Thermo Scientific Trace GC Ultra gas chromatography system that was coupled with a Thermo Scientific Trace DSQ II mass spectrometer system. An HP-1 fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) was used. The column was held at 40 °C for 2 min, then ramped to 290 °C at a rate of 4 °C/min with a final holding time of 20 min. Helium was used as a 6
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carrier gas at a constant flow rate of 1.2 mL/min. The ion source was maintained at 260 °C and operated in the electron-impact ionization (EI) mode with an electron beam energy of 70 eV. The mass range was set to m/z 50−650 and the scanning cycle was set to 100 ms. Quantification of saturated hydrocarbons was achieved on a Thermo Scientific Trace GC Ultra gas chromatography system equipped with a flame ionization detector (FID) using a deuterated internal standard (C15D32). An HP-1 fused silica capillary column (60 m × 0.32 mm i.d. × 0.25 µm film thickness) was employed with nitrogen as a carrier gas at a constant flow rate of 1.0 mL/min. The GC operation condition was identical to that used for the GC-MS analysis described above. 2.4. FT-ICR MS Analysis A Bruker Apex-ultra FT-ICR mass spectrometer equipped with a 9.4 T superconducting magnet was used to analyze the polar compounds in biodegraded oil samples. Shi et al.50,51 provided the details for sample preparation and for the ESI FT-ICR MS analysis conditions. Briefly, each of the oil samples was dissolved in toluene to produce a solution at a concentration of 10 mg/mL, and then 20 µL of each solution mixture was diluted with 1 mL of a toluene/methanol (1:3, v/v) solution. Prior to injection, 5 µL of octadecane-D35 acid (319 ng/µL) was added to serve as an internal standard. The sample solution was injected at 3 µL/min into the ESI source using a syringe pump. The ESI source was operated in the negative-ion mode. The operating conditions for negative-ion formation included a 4.0 kV spray shield voltage, a 4.5 kV capillary column front end voltage and a −320 V capillary column end voltage. Ions were accumulated in a hexapole for 0.2 s. The delay was set to 1.0 ms to transfer the ions to the ICR cell by electrostatic focusing of the transfer optics. The mass range was set to m/z 150–800 and the acquired data size was set to 4 M words. Time domain data sets were co-added 64 times to enhance the signal-to-noise ratio (S/N). 2.5. Mass Calibration and Data Analysis 7
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The mass spectra were calibrated using a known homologue series of O2 class compounds (fatty acids) with a high abundance. Peaks with a relative abundance greater than 6 times the standard deviation of the baseline noise level were exported to a spread sheet. Data analysis was performed using custom software, the details of which have been described elsewhere.52 3. RESULTS AND DISCUSSION 3.1. Composition of Saturated Hydrocarbons Characterized by GC-MS and GC Figure 1 shows the total ion chromatogram (TIC) of saturates from crude oils at various degrees of biodegradation. Normal alkanes were the homologue series that were most susceptible to biodegradation throughout the experiment, especially for those with lower carbon numbers. Isoprenoids, such as pristane and phytane, were only partially altered at week 8, as indicated by the slight variations in the ratios of pristane and phytane (Pr/Ph) and (Pr+Ph)/C30H (Table 1). This observation is consistent with that in a previous study by Sun et al.53, in which pristane and phytane became partly degraded at a moderate biodegradation stage. No significant alterations were observed for steranes and hopanes in all of the biodegraded oils. Therefore, the oils were assigned a biodegradation degree range of 1–4 on the Peters and Moldowan biodegradation scale (abbreviated as PM level 1–4).25 Preferential removal of saturated hydrocarbons prevails under both aerobic and anaerobic conditions in comparison with non-hydrocarbons. This can be attributed to the population of indigenous microbes and the bioavailability of compounds in oils as substrates. For instance, Pseudomonas sp., which are prevalent in the surrounding and often used as a degrading bacterium, has the ability to selectively utilize hydrocarbons as a sole source of carbon and energy.30,31,54–56 In addition, oil fractions display gradual changes in that the saturated fraction was consumed rapidly and the relative contents of the other fractions increased progressively (Table 1). This may indicate that the increase of resin and asphaltene fractions was in part attributed to biodegradation products of hydrocarbons. 8
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The results for the quantitative GC analysis of normal alkanes are shown in Figure 2. The n-alkanes were dominated by the carbon number range of C12–C31. Their concentrations differ from each other and also vary with increasing biodegradation. The total concentration of n-alkanes decreases gradually from 276.4 to 15.0 mg/g, with an extent of degradation up to 94.6%. Figure 2 also indicates that the susceptibility to biodegradation obviously diminishes with increasing carbon number in the range of C12–C24, but appears to be similar in the range of C25–C31 (Figure 2). This may imply that the biodegradation of n-alkanes is largely controlled by their concentrations and hydrophilicity. The n-alkanes with lower carbon numbers might facilitate access to petroleum degrading microbes as substrates due to the lower interfacial tension between the oil and the water.57 Peters et al.19 proposed that C10‒C24 n-alkanes are degraded more rapidly, while n-alkanes >C24 are more difficult to transport across cell membranes and n-alkanes
