Effects of Alkyl Chain Branching on the Biotransformation of

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Environ. Sci. Technol. 2008, 42, 9323–9328

Effects of Alkyl Chain Branching on the Biotransformation of Naphthenic Acids B E N J A M I N E . S M I T H , †,§ C. ANTHONY LEWIS,† SIMON T. BELT,† CORINNE WHITBY,‡ AND S T E V E N J . R O W L A N D * ,† Petroleum Environmental Geochemistry Group, School of Earth, Ocean and Environmental Sciences, University of Plymouth, PL4 8AA, U.K., and Department of Biological Sciences, Wivenhoe Park, University of Essex, CO4 OLH, U.K.

Received July 11, 2008. Revised manuscript received October 2, 2008. Accepted October 15, 2008.

The rapid expansion of the oil sands industry has seen a concomitant expansion of the production of associated waste containing toxic naphthenic acids (NAs). Bioremediation of such waste is thus an important goal, but the mechanisms of biodegradation are still poorly understood, despite recent advances. Many oil sands NAs are resistant to biodegradation, and alkyl side chain branching has been invoked as an explanation. To investigate this hypothesis we examined the biotransformation by a sedimentary bacterial community of novel, synthetic, surrogate NAs (butylcyclohexylbutanoic acids (BCHBAs)) with variously branched butyl side chains (n- through t-) and unbranched alkanoate groups, plus one (4-(4′isobutylcyclohexyl)pentanoic acid (iso-BCHPA)), with both branchedbutylandbranchedalkanoatechains.Sedimentmicrobial populations were inoculated into media containing the individual surrogate NAs, and gas chromatography-mass spectrometry (GC-MS) was used to determine the extent of biotransformation. Biotransformation decreased as NA side chain branching increased. For example, over 97% of the n-BCHBA with the nonbranched alkyl side chain was transformed in 30 days compared to the tert-BCHBA with the most highly branched side chain where only 2.5% was transformed. Both the iso-BCHBA and sec-BCHBA had intermediate transformation rates with about 77% and 47% transformed respectively after 30 days. The metabolites were identified as butylcyclohexylethanoic acids in each case, indicating beta-oxidation of the alkanoate substituents. The iso-BCHPA with both chains branched was resistant to degradation. The results are thus consistent with earlier hypotheses for the resistance of oil sands NAs. Identification of bacteria capable of oxidizing such branched alkyl chains-or of attacking the cyclic rings of NAs, may be important if bioremediation of oil sands NAs is to be achieved.

* Corresponding author phone: +44 1752 233013; fax: +44 1752 233035; e-mail: [email protected]. † University of Plymouth. ‡ University of Essex. § Present address: Oil Plus Ltd., Dominion House, Kennet Side, Newbury, Berkshire, RG14 5PX, U.K. 10.1021/es801922p CCC: $40.75

Published on Web 11/12/2008

 2008 American Chemical Society

Introduction Naphthenic acids (NAs) are complex mixtures of alkyl substituted, mainly alicyclic, carboxylic acids fitting the general formula CnH2n+zO2, where z denotes the hydrogen deficiency resulting from ring formation. For example, monocyclic acids have z ) -2, bicyclic acids z ) -4 and so on (1). NAs occur particularly in heavy, immature, crude oils and in oil (tar) sands (reviewed in refs 1-3). The acids can cause severe exploration, environmental, and engineering problems due to their toxicity, corrosiveness and tendency to form metal salt deposits (e.g., refs 4-7). With the sustained high price of crude oil, it is becoming increasingly viable to produce oils with high NA contents (6, 7). For example, Canada’s oil sands contain very high NA concentrations and are destined to be the main supply of foreign oil to the U.S. for the next century. It is predicted that in less than 20 years the amount of NA-contaminated waste produced globally will exceed a billion m3 (1-3, 8). NAs are among the most toxic components of both refinery effluent and oil sands tailing waters (1). This toxicity has often been associated with their surfactant properties (4, 5). In fact numerous studies have shown that NAs are toxic to a wide variety of organisms including mammals, fish, and zooplankton (1, 2, 9-12) at aqueous concentrations as low as 100 ppb (µg L-1) and some are also suspected endocrine disruptors (2). Therefore the microbial degradation of NAs is potentially very important for reducing both the amount and associated toxicity of NAs in the environment (1). Many early studies of NA biodegradation were hindered by a lack of knowledge of detailed NA chemical structures. This resulted largely from a deficit of analytical methods which were sufficiently discriminatory to resolve the complex NA mixtures. Recent advances in chromatography and mass spectrometry have remedied this somewhat (e.g., refs 13-15). Early evidence suggested that NAs contain both alkyl and alkanoic acid groups (1, 16-18) but few, if any, appropriate acids containing both these alkyl and alkanoate side-chains for detailed mechanistic studies have ever been available from commercial sources (1, 2, 17, 19) and the lack of available substrates has hindered progress in identifying which components of a microbial community are capable of NA degradation. Understanding the processes by which microorganisms degrade the different structural regions of NAs is vital (1). For example, Scott et al. (20) showed that commercial NAs were degraded more readily than oil sands NAs, suggesting the structures were different, while Quagraine et al. (2) also showed that the recalcitrance of some NAs is indeed related to their structures and that the more recalcitrant NAs are those with alkyl substituted aliphatic chains, tertiary substitution at positions other than the β-position to the carboxylic acid of the main carbon chain, methyl substitution on rings and cis-isomerism in alicyclic acids. Several studies using commercially available surrogate NAs have been made, but these compounds may not be the most environmentally relevant models (17, 18, 21, 22). More recent studies have led to suggestions that the NAs which are most resistant to biodegradation indeed have branched alkyl chains (3, 15), but again examples of such acids were not commercially available. The aim of the present study was therefore to synthesize a series of C14 monocyclic acids possessing both C4 alkyl and C4 or C4 methyl substituted alkanoate substituents and to investigate which structural features influenced the extent of biotransformation of the NAs. The branching in the alkyl substituent was varied in a systematic way from n-butyl (nVOL. 42, NO. 24, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Synthetic routes and structures of surrogate NAs. a. AlCl3 b. KOH/N2H4 c. H2/HAc/PtO2, ∼ 8 psi. Details of syntheses are given by Smith (23). Both cis- and trans- diastereomers were produced by the hydrogenation step c. BCHBA; least branched; Figure 1) through sec-butyl (secBCHBA), iso-butyl (iso-BCHBA), and tert-butyl (tert-BCHBA; most branched; Figure 1). The carbon number (C14) was chosen to be fairly typical of NAs in oils and oil sands (e.g., ref 3). All four surrogate NAs were also selected on the basis of their toxicity (23). In addition we also studied the biodegradation of one C15 NA, (4-(4′-isobutylcyclohexyl)pentanoic acid, (iso-BCHPA)) which contained both branched butyl and alkanoate chains (Figure 1). We believe the results demonstrate an advancement in existing knowledge into the stepwise biodegradation of monocyclic NAs and provide important information on the operation of NA degradation pathways. This will enable future research into the elucidation of the functional genes involved in NA degradation which would be an advance toward the ultimate goal of bioremediation of these environmental pollutants.

Experimental Section Synthesis of Surrogate Naphthenic Acids. The surrogate NAs n-BCHBA, iso-BCHBA, sec-BCHBA, tert-BCHBA, and isoBCHPA were synthesized by the routes outlined in Figure 1. Brief details of the syntheses are reported in the Supporting Information, and full details of the syntheses and characterization of intermediates and final products by spectroscopic methods, are reported by Smith (23). Environmental Sampling. Sediment samples (50 g) used as a source of bacterial inocula were obtained in January 2003, from Devonport, Tamar estuary, Devon (U.K.) (50:22: 36N, 4:10:53W) using 50 mL sterile disposable plastic centrifuge syringes. The upper 0-3 cm was taken. Sediments were transported to the laboratory at 4 °C within 2 h of sampling and stored at -20 °C. The sediments were black/ brown in color, the pH was 7.5 and the total organic content (TOC) 1.6% of dry weight after removal of inorganic carbonate by HCl. The grain size was 125-500 µm, determined by wet sieving through 2000, 1000, 500, 125, and 90 µm sieves. The site chosen is adjacent to a busy ferry port and naval dockyard which has operated for 300 years and the sediments have been subject to periodic hydrocarbon exposures typical of an industrial dockyard, in modern times. (The sediments in this area have been the subject of numerous previous studies over the last 120 years due in part to the proximity of the laboratories of the UK Marine Biological Association, built 9324

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1888 and the Plymouth Marine Laboratory). Comprehensive reviews of the science of the estuary, including of analyses of polycyclic aromatic hydrocarbons and pesticide residues in the sediments appeared in 2003 (24) and prior to proposed dredging activities in 2007 (25, 26). Composition of Inoculum Bacterial Community and Enrichment of Na-Degrading Bacterial Communities. The composition of the bacterial community of the sediments as an inoculation source of bacteria was briefly studied by wellaccepted molecular biological techniques including DNAtargeted PCR amplification of the 16S rRNA genes followed by DGGE analysis and generation of 16S rRNA gene clone libraries (e.g., ref 27-31,) using DNA extracted from the original sediment (inoculum). Selected clones and predominant bands from the DGGE gels were sequenced and the 16S rRNA gene sequences which were recovered demonstrated close sequence identity to the β-Proteobacteria (genera Alcaligenes (99%) and Achromobacter (99%)). Both of these genera have been implicated in the biodegradation of hydrocarbons (32). Furthermore, Alcaligenes sp. has also been shown to biodegrade naphthalene from bitumen in tar sands (33). Additional 16S rRNA gene sequences which were recovered from the DGGE gels and clone libraries demonstrated a close sequence identity to the γ-Proteobacteria (genera Pseudomonas (97%) and genera Stenotrophomonas (98%)). There are numerous studies in the literature demonstrating the catabolic ability of Pseudomonas spp. for organic substrates. For example, a study by Ro¨ling et al. (34) demonstrated the predominance of sequences relating to Pseudomonas sp. during initial stages of oil degradation. Starter cultures were prepared using sediment (1 g) inoculated into 50 mL minimal medium containing (g L-1): MgSO4, 0.2; (NH4)2SO4, 0.5; KH2PO4, 0.5; K2HPO4, 1.5; NaOH, 0.02; Na2 EDTA, 0.12; ZnSO4, 0.004; Cu SO4, 0.001; Na2 SO4, 0.0001; Na2 MoO4, 0.001; MnSO4 0.0004; CoCl2, 0.0001, (pH 7.0) and supplemented with the surrogate NAs: n-BCHBA, sec-BCHBA, and decahydronaphthoic acid (naphthoic acid hydrogenated in-house; purity >90%). Bottles were crimp sealed using PTFE seals to prevent volatilisation of the hydrocarbons (Supelco). The degradation experiments were prepared by inoculating 1 mL of the Devonport sediment starter culture in triplicate flasks containing 25 mL minimal media supplemented with 50 µL (0.2 mg) of either n-BCHBA, sec-BCHBA, iso-BCHBA, tert-BCHBA, or iso-BCHPA dissolved in methanol (HPLC grade, Rathburn Chemicals Ltd., Scotland; final concentration 4 mg mL-1). Flasks were crimp sealed and incubated in the dark at 25 °C. Subsamples were removed at 0, 1, 3, 9, 14, 21, and 30 days (42d for iso-BCHPA) for analysis. Abiotic controls and procedural blanks containing the media and NAs, but no bacterial inoculum were also analyzed under the same conditions to assess and account for possible losses due to sorption or volatilisation (23). Surrogate NA Extraction and Gas Chromatography Analysis. Triplicate flasks containing 1-adamantecarboxylic acid (99%; Aldrich, UK) and icosane (n-C20; 99%; Aldrich, UK) dissolved in methanol (final concentration 1 mg mL-1) were used as internal standards. Samples (25 mL) were removed from the enrichment cultures and centrifuged at 4000g for 10 min. The supernatant was transferred to a glass Quickfit boiling tube (50 mL), acidified to pH 2 (concd HCl) and extracted with ethyl acetate (3 × 15 mL). Extracts were combined, dried (Na2SO4; overnight) and evaporated to neardryness (Buchi, 40 °C), transferred to GC vials and gently blown down to dryness (N2). BSTFA (20 µL) was added to each sample and samples heated (70 °C; ca. Twenty min). Samples were resuspended in dichloromethane (1 mL) and examined using gas chromatography-mass spectrometry (GC-MS) comprising a HP5890 Series II GC (Hewlett-Packard, U.S.) fitted with a HP5970 series Mass Selective Detector and HP7673 autosampler. GC-MS conditions were 1 µL splitless

FIGURE 2. Biotransformation of NAs by the Devonport consortia expressed as the percentage of each NA recovered versus time. injection; injector temperature 250 °C; column Agilent Ultra-1 (12.5 m × 0.20 mm × 0.33 µm); GC program, 40-300 at 5 °C min-1, hold 10 min; head pressure 70 kPa; electron multiplier, 1600 V. Quantification was performed by comparing analyte peaks areas to those of standards.

Results and Discussion Synthesis and Characterization of NAs. The surrogate NAs containing straight chain and branched butyl substituents and not available previously (viz: n-BCHBA, iso-BCHBA, secBCHBA, tert-BCHBA) were each successfully synthesized with >94-99% purity (GC-MS of TMS esters) by the use of traditional Friedel-Crafts acylation routes involving coupling of the respective butylbenzenes with succinic anhydride, reduction of the resulting keto groups of the keto-acid (traditional Huang-Minlon chemistry), and hydrogenation, as outlined in Figure 1. All intermediates were characterized by infrared (IR) and 13C and 1H nuclear magnetic resonance (NMR) spectroscopy in addition to GC-MS (23). Each final alicyclic acid product comprised a mixture of cis- and transdiastereomers (major, first-eluting, isomers 60-70%, minor, second-eluting, isomers 30-40%), as expected from the final nonstereospecific hydrogenation step. These were separable by GC-MS as the TMS esters and the isomers of each pair had virtually identical mass spectra to one another. Preparative separation and thus assignment, of the elution order of the isomers, was not possible and NMR spectroscopy was carried out on each diastereomeric pair as a mixture (23). Synthesis of iso-BCHPA (Figure 1) employed similar chemistry to that used above, but γ-valerolactone was used in place of succinic anhydride. This synthesis produced a 26:42:32% mixture of the o-, m-, and p- butylphenylpentanoic acids from which the pure p- isomer was isolated by preparative HPLC (23). Hydrogenation produced the alicyclic acid (>96% purity as TMS ester), again as a mixture of cisand trans- diastereomers. The mass spectra and chromatographic properties of these synthetic acids (Supporting Information) will be useful for comparison with those of natural oil and oil sands NAs (cf. 1-3). The individual compounds were used in a series of incubations with bacteria to investigate the influence of alkyl and alkanoate branching on the biotransformation of NAs. Biotransformation of Synthetic NAs. The mean percentage recoveries of triplicate analyses of four surrogate NAs (n-BCHBA, iso-BCHBA, sec-BCHBA, and tert-BCHBA) during 30 days incubation with a microbial consortium from Devonport sediment are presented in Figure 2. The abiotic

controls for each surrogate showed that any reduction in surrogate NA concentrations was due to biotic transformation. There was a clear relationship between the extent of biotransformation and branching of the alkyl substituents over the 30 day incubation period (Figure 2). The highly branched tert-BCHBA was the most difficult to transform by the Devonport consortium with only at most 2.5% transformed after 30 days. The greatest extent of biotransformation was observed with n-BCHBA with over 97% transformed at day 9. The iso-BCHBA and the sec-BCHBA exhibited intermediate effects with 77 and 47%, respectively, transformed following 30 days incubation. Some differences in the extent of biotransformation of the two GC-MS peaks representing diastereomeric (cis- and trans-) acids of each of the susceptible surrogate NAs (n-BCHBA, iso-BCHBA, sec-BCHBA, and tert-BCHBA) were also observed. The individual diastereomers in each GC-MS peak could not be assigned herein, but in each case the less abundant (33, 34, 40, 43% of the total for n, iso, sec, tert-BCHBA, respectively), second-eluting (GC-MS) diastereomer was degraded fastest. The relative susceptibility to biotransformation was thus n- > iso- > sec> tert-BCHBA with the n-BCHBA diastereomer completely transformed between 3-9 days and the iso-BCHBA between 9 and 14 days; whereas the diastereomer of sec-BCHBA had been reduced from 40 to 8% of the total by day 30 and the tert-BCHBA diastereomer only reduced from 43 to 31% by day 30. This is a further indication of the importance of structure (in this case the stereoisomerism about the cyclohexyl ring) on the biotransformation rates of NAs. Differences in the biodegradation of cis- and trans- isomers of cyclic NAs have been observed previously, with the cis- isomer generally more resistant (35). (Whether this implies that the GC-MS elution order of the present synthetic acids was cis(more resistant), before trans- (less resistant), is a moot point). Interestingly, Peng et al. (36), showed that, in contrast to the differences in biodegradation observed for these isomers previously (35) and now herein (vide infra), the adsorption coefficients (Kd) of cis- or trans- isomers of 4-methylcyclohexylethanoic acid and 4-methylcyclohexanecarboxylic acid onto sediments were unaffected by structure. This indicates, perhaps, that differential adsorption onto sediments and hence controlled accessibility of the substrates is not an explanation for the differences in biotransformation rates of the cis- and trans- isomers. Indeed, the low Kd values suggest that these monocyclic NAs would partition mainly into the water column (36). VOL. 42, NO. 24, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Biotransformation of 4-(4′-isobutylcyclohexyl)butanoic and 4-(4′-isobutylcyclohexyl)pentanoic acids by the Devonport consortium expressed as the percentage of each NA recovered versus time. Bars represent ( standard deviations (n ) 3). When an acid incorporating a further branch point, this time in the alkanoate substituent, was synthesized herein (iso-BCHPA; C15; Figure 1) and incubated with the initial Devonport starter consortium (but which had been held in culture for some extra months), no biotransformation was observed- even after 42 days, compared to more than 20% alteration of the C14 iso-BCHBA reincubated at the same time (Figure 3). This result thus provides evidence that the lower extent of transformation is the result of the increased branched structure in the alkanoic acid side chain, as postulated recently (3). Transformation of the C14 iso-BCHBA was slower in this repeat experiment than previously (cf. Figures 2 and 3) which is probably due to changes in the starter culture during storage. This indicates the value of a concomitant reincubation of BCHBA with the BCHPA. It is clear from the combined results of the present and previous studies (3, 23) that important controlling factors for the biodegradability of NAs thus include increasing cyclization (3), increasing branching in alkyl and alkanoate substituents (vide infra) and the stereochemistry of the substituted alicyclic rings (ref 35; vide infra). Of course there may be many differences between the biodegradation of pure synthetic NAs compared to natural NAs in oil sands and in refinery mixtures, and the use of a small amount of methanol solvent herein to dissolve the synthetic NAs may mean that the NAs are degraded by cometabolism, while the methanol was used as an energy source. However, it is worth noting perhaps, that for n-BCHBA (n ) 14, Z ) 2) for example, the 97% biotransformation in 9 days in the present experiments (Figure 2) was within the range observed by Han et al. (3) for biodegradation of comparable acids in mixtures of refined (Merichem) NAs (80% in 6 days for n ) 14, z ) 2). Metabolite Production from Biotransformation of Synthetic NAs. The increase in metabolite production by the Devonport consortium during degradation of four of the surrogate NAs for which some transformation was observed, was also determined by GC-MS (Figure 4). The extent of transformation of the NAs correlated broadly with an increase in the production of metabolites. The n-BCHBA produced the highest proportion of metabolites (relative to the initial NA), followed by the iso-, sec-, and tert-BCHBA (Figure 4). 9326

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Gas chromatograms of the surrogate NAs at day 30 demonstrated the presence of major (Figure 5A-D) and minor metabolites (diastereomers of the major metabolic products). The mass spectra of each of the major metabolites were all very similar, each with a base peak ion of m/z 117, due to a fragmentation characteristic of TMS ester derivatives, and molecular ions of m/z 270, corresponding to molecular weights of 198 if the metabolites were underivatised acids (Figure 5A-D). Since the putative molecular weight of the metabolites was 28 mass units less than that of the surrogate NAs, the proposed identities of the major metabolites are 4-(4′-n-butylcyclohexyl)ethanoic acid, 4-(4′-iso-butylcyclohexyl)ethanoic acid, 4-(4′-sec-butylcyclohexyl)ethanoic acid, and 4-(4′-tert-butylcyclohexyl)ethanoic acid (n-BCHEA, isoBCHEA, sec-BCHEA, and tert-BCHEA). However, these cannot be confirmed without comparing the retention times and mass spectra to those of authentic compounds which are not commercially available and require synthesis. However, the proposed identities are consistent with the known mechanisms of aerobic biodegradation of aliphatic and alicyclic acids (refs 3, 37, and references therein). By the most common route of β-oxidation, cyclohexylalkyl acids with even carbon number alkanoate substituents (e.g., butanoate herein) are expected to degrade to cyclohexylethanoic acid (2, 37). Microbes able to degrade the latter further by R-oxidation are not widely distributed (37). The presence of the butylcyclohexylethanoic acid metabolites in each case shows that oxidative attack was actually on the alkanoate substituent of the original NAs and did not take place on the alkyl side-chains, despite the fact that the alkyl branching effected the rate of degradation (Figure 2). The mass spectra of the derivatized minor metabolites detected in each case for the n-, iso-, and sec-BCHBAs, exhibited molecular ions of 268 (as TMS esters), corresponding to a molecular weight of 196 for underivatised acids. These metabolites were thus tentatively assigned as monounsaturated derivatives of n-BCHEA, iso-BCHEA, and sec-BCHEA, which is also consistent with a β-oxidation desaturase step in the further degradation of the BCHEAs (ref 2 and references therein). These results suggest that the most resistant NAs in, for example, oil sands might include significant proportions of

FIGURE 4. Percentage production of major metabolites (butylcyclohexylethanoic acids) during biotransformation of the surrogate NAs by the Devonport consortium over 30 days. Error bars represent ( standard deviations (n ) 3); recovery is expressed as a percentage of the initial amount of substrate.

FIGURE 5. (A) Mass spectrum of major metabolite formed from biotransformation of n-BCHBA (as TMS ester). (B) Mass spectrum of major metabolite formed from biotransformation of iso-BCHBA (as TMS ester). (C) Mass spectrum of major metabolite formed from biotransformation of sec-BCHBA (as TMS ester). (D) Mass spectrum of major metabolite formed from biotransformation of tert-BCHBA (as TMS ester). branched alkylcyclohexylethanoic acids and branched alkyl polycycloethanoic acids. Interestingly, this was indeed demonstrated recently when a novel amide derivatization and LC-MS method was used to characterize such mixtures (23, 39). In combination with those analytical data the results herein therefore suggest that a reduction in the toxicity of the more bioresistant NAs in oil sands (e.g., branched alkyl polycycloethanoic acids) may require microorganisms capable of oxidizing the branched alkyl groups, ethanoate groups or alicyclic rings of NAs.

Clearly the key microbial species involved in NA degradation have yet to be isolated and cultured. This might be a useful target for future studies now that more relevant synthetic surrogate NAs are available and a better understanding of the effects of molecular structure (specifically branching in alkyl and alkanoate substituents), has emerged.

Acknowledgments We thank the University of Plymouth for a PhD halfstudentship (BES). VOL. 42, NO. 24, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Supporting Information Available Details of the synthetic methods for the acids and electron impact mass spectra of the trimethylsilyl esters of the four synthetic butylcyclohexylbutanoic acids are provided. This material is available free of charge via the Internet at http:// pubs.acs.org.

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