Article pubs.acs.org/est
Effect of Alkyl Side Chain Location and Cyclicity on the Aerobic Biotransformation of Naphthenic Acids Teresa M. Misiti,†,§ Ulas Tezel,†,‡,§ and Spyros G. Pavlostathis*,† †
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0512, United States The Institute of Environmental Sciences, Bogazici University, Istanbul 34342, Turkey
‡
S Supporting Information *
ABSTRACT: Aerobic biodegradation of naphthenic acids is of importance to the oil industry for the long-term management and environmental impact of process water and wastewater. The effect of structure, particularly the location of the alkyl side chain as well as cyclicity, on the aerobic biotransformation of 10 model naphthenic acids (NAs) was investigated. Using an aerobic, mixed culture, enriched with a commercial NA mixture (NA sodium salt; TCI Chemicals), batch biotransformation assays were conducted with individual model NAs, including eight 8-carbon isomers. It was shown that NAs with a quaternary carbon at the α- or β-position or a tertiary carbon at the β- and/or β′-position are recalcitrant or have limited biodegradability. In addition, branched NAs exhibited lag periods and lower degradation rates than nonbranched or simple cyclic NAs. Two NA isomers used in a closed bottle, aerobic biodegradation assay were mineralized, while 21 and 35% of the parent compound carbon was incorporated into the biomass. The NA biodegradation probability estimated by two widely used models (BIOWIN 2 and 6) and a recently developed model (OCHEM) was compared to the biodegradability of the 10 model NAs tested in this study as well as other related NAs. The biodegradation probability estimated by the OCHEM model agreed best with the experimental data and was best correlated with the measured NA biodegradation rate.
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INTRODUCTION Naphthenic acids (NAs) are a complex group of alkylsubstituted acyclic, monocyclic, and polycyclic carboxylic acids.1−3 Classic NAs have the general formula CnH2n+ZO2, where n is the carbon number and Z is the number of hydrogen atoms lost due to ring formation, also known as hydrogen deficiency. Z is zero or a negative, even integer, and reflects the degree of cyclicity. Recently, use of advanced mass spectrometry techniques has shown that oil sands process-affected water (OSPW) and oil field and refinery wastewaters contain low levels of NAs with a broad spectrum of molecular structures.4−9 The range of NA structures has recently been expanded to include other polar and N- and S-containing heteroatomic species and aromatic species found in the oil sands acid extractable fraction.5,10−15 NAs in the wastewater is an ecological concern, as NAs are among the most toxic components of both OSPW and refinery process wastewater. The degree of toxicity depends on the concentration of NAs as well as their type and molecular structure.1,16−25 Many NAs are biodegradable, and development of effective biological processes could play an important role in the remediation of NA-affected environments, such as tailings ponds holding OSPW and oil refineries wastewaters. Biodegradation of NAs commences with β-oxidation that results in mineralization with the formation of carbon dioxide.26 © 2014 American Chemical Society
However, the biodegradability and the rate of biodegradation of NAs are related to the molecular structure. Analytical limitations have made it difficult to identify specific structures in complex NA samples; however, commercially available model NAs have been used to investigate the effect of structure on NA biodegradation. Under aerobic conditions, the rate and extent of biodegradation was lower for NAs with higher cyclicity (i.e., high Z value) and alkyl branching.27−30 In general, studies investigating the effect of NA structure on biodegradability have shown that the more complex NAs, i.e., those with higher cyclicity, branching, and molecular weight (MW), are the most recalcitrant.27−29,31−37 It has been suggested that a quaternary carbon adjacent to a carboxylic group or a tertiary carbon at the β-position contribute to the recalcitrance of NAs.27 Although previous studies have investigated the effect of cis/trans isomerism38,39 and branching of the alkyl side chain28 on the biodegradation of some simple NAs, information on the effect of structure on the biodegradability of NAs is relatively limited. In addition, few studies have correlated NA structure to biotransformation Received: Revised: Accepted: Published: 7909
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Biotransformation Assays. Aerobic batch assays were performed with the 10 model NAs using an aerobic, NAenriched culture. The culture was developed with refinery activated sludge mixed liquor inocula, enriched and maintained with the NA mixture from TCI Chemicals as the sole carbon and energy source for over 3 years. The development, maintenance, and molecular characterization of the NAenriched culture have been previously described.21 The TCI NA mixture is representative of the types of NAs found in refinery wastewater streams, having a similar NA congener distribution to refinery desalter brine, which was identified as the main source of NAs in refinery wastewater.20 Thus, use of this culture in the work presented here, which is related to the biodegradation of NAs in refinery wastewater, was an appropriate choice. Aliquots of the NA-enriched culture were washed three times with 10 mM phosphate buffer until the total residual NA concentration was less than 3 mg/L and then resuspended in culture medium containing the following (in g/ L): K2HPO4, 1.07; KH2PO4, 0.524; CaCl2·2H2O, 0.068; MgCl2·2H2O, 0.135; MgSO4·7H2O, 0.268; FeCl2·4H2O, 0.068; 0.67 mL/L trace metal stock solution and amended with NH4Cl (20 mg N/L).21 In order to assess the rate and extent of biotransformation of the 10 model NAs, an aerobic biotransformation assay was conducted as follows. Portions of 100 mL of the washed and resuspended culture were added to 10 250-mL Erlenmeyer flasks (200 mL liquid volume), and each model compound was added at an initial concentration of approximately 50 mg/L (ca. 350 μM) and the pH was adjusted to 7 using 1 N HCl. An abiotic control series was also set up in the same manner using only culture medium, 20 mg NH4+-N/L and each model NA at 50 mg/L (ca. 350 μM). The initial biomass concentration was 305 ± 19 mg of volatile suspended solids (VSS)/L. The flasks were loosely capped and agitated on an orbital shaker at 190 rpm at room temperature (22−24 °C). Total NAs and pH were measured throughout the incubation period. A closed bottle, aerobic biotransformation assay was conducted with the NA-enriched culture to determine to what extent two model compounds, valproic acid (n = 8, Z = 0) and cyclohexylacetic acid (n = 8, Z = −2), were mineralized to CO2. These compounds were chosen among all 8-carbon NAs representing acyclic and monocyclic model NAs. The NAenriched culture biomass was washed, resuspended in culture medium, and amended with 20 mg NH4+-N/L. The washed culture was then divided into three culture series as follows: two duplicate sacrificial culture series were set up in 25 mL serum bottles amended with 15 mL of NA-enriched culture and 50 mg NA/L of each model NA, two duplicate sacrificial control series were set up with 50 mg/L (ca. 350 μM) of each model NA and 200 mg/L sodium azide to inhibit any biological activity, and a duplicate control culture series was set up with only the NA-enriched culture and no NAs. All bottles were sealed with Teflon-lined stoppers and aluminum crimps and then 2 mL of pure O2 was injected into the headspace containing air to maintain a positive pressure and to ensure oxygen was in excess. The initial biomass in all serum bottles was 219 ± 5 mg VSS/L. The culture series were incubated at room temperature (22 to 24 °C), agitated on an orbital shaker at 190 rpm. On day 0, 1, 2, 3, and 7, each culture series was sacrificially sampled for headspace CO2 and O2, total NAs, soluble chemical oxygen demand (COD), volatile fatty acids (VFAs), organic acids, pH, and possible intermediates. On each sampling day, two NA-amended and two NA-free culture
kinetics. The objective of this study was to evaluate the effect of the alkyl chain location and cyclicity on the aerobic biodegradation and kinetics of model NAs, using both biodegradation assays and quantitative structure−activity relationships (QSAR) in order to improve our understanding of NA recalcitrance.
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MATERIALS AND METHODS Chemicals. The work reported here is part of a broader study that has focused on classical NAs in the context of oil refineries.19−21 Ten model NA compounds were chosen (Table 1) on the basis of the following rationale. Eight compounds Table 1. Structural Properties and Gibbs Free Energy of Formation (ΔG0f ′) and Oxidation (ΔG0r ′) of Select Model NAs Used in This Study
with the same carbon number (n = 8) were used to compare the aerobic biodegradation of aliphatic and monocyclic NAs (Z = 0 vs −2) considering alkyl branching and its position; two compounds (DAA and 5β-CA) were also tested as examples of di- and tetracyclic NAs (n = 14, Z = −4 and n = 24, Z = −8, respectively). All ten carboxylic acids were purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions (5−10 g/L) of each NA were made in 1 N NaOH. NA solutions used in the assays were adjusted to pH 7 with 1 N HCl. Consistent with the focus of our broader study on oil refineries, in particular activated sludge treatment systems, all biodegradation assays were conducted at circumneutral pH values. A commercial mixture of NA sodium salt was purchased from TCI Chemicals (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The characteristics of the TCI NA mixture have been previously reported.19−21 7910
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(OCHEM),44 which combines the databases used by the BIOWIN models, the Chemical Risk Information Platform (CHRIP) from the Japanese MITI database, and the European Chemical Agency (ECHA) database. All biodegradation models estimate biodegradation probability on the basis of certain descriptors and molecular fragments. Compared to the BIOWIN models, OCHEM uses a more detailed QSAR model developed with various machine learning methods. Biodegradation probability values equal to or above 0.50 indicate biodegradable compounds. Analytical Methods. COD, VSS, and pH were determined following procedures outlined in Standard Methods for the Examination of Water and Wastewater.45 Total gas and gas composition were measured with a pressure transducer and by gas chromatography/thermal conductivity detection, respectively. Nonflame ionizable organic acids were measured with a high-performance liquid chromatography unit equipped with UV/visible diode array and refractive index detectors. The samples were centrifuged, and the supernatant was acidified with 0.2 N H2SO4 in a 1:1 volume ratio and filtered through 0.2 μm membrane filters before the analysis. VFAs (C2−C7) were measured by gas chromatography with flame ionization detection. NA concentrations were determined using a pair-ion extraction method, followed by liquid chromatography/mass spectrometry (LC/MS; direct infusion) as previously described.20 p-Toluene sulfonate (pTS) was used as a surrogate standard. The extraction efficiency of NAs from culture samples was above 95%, and no interference of the LC/MS method with the culture medium was observed. The MS had a resolution of 0.2 m/z and a mass accuracy of two significant figures. Possible intermediates formed from the degradation of the model NAs were monitored using an LC/MS scanning m/z range from 30 to 1000 in both positive and negative electrospray ionization (ESI) mode.
bottles were acidified with 1 mL of 6 N H2SO4 and agitated for 4 h, and the headspace CO2 concentration was measured in triplicate. Prior to opening for liquid analysis, the headspace O2 concentration in the nonacidified serum bottles was quantified. The sodium azide-amended series was analyzed for all of the above-mentioned parameters at the end of the 7-day incubation period. The extent of NA mineralization was determined on the basis of carbon mass balance calculations. Biomass incorporated carbon in the NA-amended culture series was determined by assuming C5H7O2N as the biomass molecular formula.21 Modeling and Simulation of NA Biotransformation. Monod kinetics were used to describe substrate (i.e., NA) utilization and microbial growth for each individual model NA as follows ⎛ kNAS NA ⎞ dS NA =−⎜ ⎟XNA dt ⎝ KSNA + S NA ⎠
(1)
⎛Y k S ⎞ dXNA = ⎜ NA NA NA ⎟XNA − bXNA dt ⎝ KSNA + S NA ⎠
(2)
where SNA and XNA are NA and biomass concentrations (μmol NA/L and mg VSS/L, respectively), t is time (days), kNA is the maximum specific biodegradation rate (μmol NA/mg VSS day), KSNA is the half-saturation constant (μmol NA/L), YNA is the microbial yield coefficient (mg VSS/μmol NA), and b is the microbial decay coefficient (day−1). On the basis of Gibbs free energy values calculated in this work (see below) and bioenergetic calculations following the procedure described in Rittmann and McCarty,40 the yield coefficient for the aerobic degradation of the model NAs was calculated as 0.42 mg VSS/mg NA-COD (equal to 0.141 mg VSS/μmol NA for NAs with a MW equal to 144.2 g/mol and 0.148 mg VSS/μmol for NAs with a MW equal to 142.2 g/ mol). The initial biomass concentration, XNA, was 305 mg VSS/ L. The decay rate values for heterotrophs are generally in the range of 0.05−0.15 day−1.40,41 Varying the decay rate value between 0.05 and 0.15 day−1 in numerical simulations did not affect the estimated maximum specific biodegradation rate, kNA; thus, a microorganism decay rate of 0.1 day−1 was chosen and kept constant for all simulations. The system of the two differential equations (eqs 1 and 2, above) was solved numerically with the Runge−Kutta method and fitted to the experimental data by error minimization to estimate the individual NA maximum specific biodegradation rate (kNA) using the Igor Pro v. 6.03 FitODE package (WaveMetrics, Lake Oswego, OR). The standard error of the estimates was calculated using the χ2 test. Estimation of NA Biodegradation Probability. The NA molecular structure was used as the descriptor and the aerobic biodegradation probability was calculated using QSAR models. ChemDraw v.13 (Cambridge Soft Inc., Cambridge, MA) was used to obtain simplified molecular input line entry system (SMILES) notations of the model NAs tested. SMILES were then used as input to OECD QSAR Toolbox42 version 2.3 to estimate the biodegradation probability of select NAs with two widely used models: BIOWIN 2, a nonlinear biodegradation probability model based on the Syracuse BIODEG chemical database, and BIOWIN 6, a nonlinear biodegradation probability model based on the Japanese Ministry of International Trade and Industry (MITI) chemical database.43 In addition, biodegradation probability estimates were obtained using the Online CHEmical Modeling Environment
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RESULTS AND DISCUSSION NA Biotransformation Potential and Kinetics. The standard Gibbs free energies of formation (ΔG0f ′) of NAs have only been reported for representative classes of NAs in the commercial TCI NA mixture.46 The standard Gibbs free energy of formation, calculated by the group contribution method developed by Mavrovouniotis,47 and the free energy of reaction for the oxidation of the NAs used in this study are shown in Table 1. The Gibbs free energy of formation (kJ/mol) decreased (i.e., became more positive) as the number of carbons and rings increased, thus leading to a lower free energy per mole. The reduction half-reactions and oxidation reactions for the model NAs are shown in Tables S1 and S2 (Supporting Information), respectively. The Gibbs free energy of reduction half-reaction varied from 27.6 kJ/electron equivalent (e−eq) (n = 24, Z = −8) to 29.2 kJ/e−eq (n = 8, Z = −2). The free energy of NA oxidation was between −4531.7 kJ/mol (n = 8, Z = −2) and −14 032.1 kJ/mol (n = 24, Z = −8). Therefore, aerobic biodegradation of all model NAs is energetically feasible, and complete NA mineralization should yield on average −107.3 kJ/e−eq. These free energy values are comparable to those calculated for the aerobic biodegradation of NAs with n = 17 and Z values from 0 to −8 (−106.9 to −106.3 kJ/e−eq).46 The pH during the aerobic biotransformation assay was maintained between 6.5 and 7 in all culture series. The NA concentration in the controls did not change in the course of incubation (data not shown), suggesting that any disappearance 7911
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days (Figures 1 and S1, Supporting Information). Lag periods were observed for the two branched, acyclic NAs (2-EA and VA). The lag period for 2-EA was approximately 1 day, upon which degradation proceeded rapidly, while for VA the lag period was less than 1 day; however, once VA degradation commenced, its degradation was much slower compared to that of 2-EA (Table 2). NA biodegradation was simulated using Monod kinetics, and kNA values were estimated as mentioned above. Before the estimation of the kNA values for each NA, the KSNA value for the degradation of 4M1-CCA was estimated because this data set had the highest degrees of freedom (i.e., number of data points >0), leading to a more precise KSNA estimation. The resulting KSNA value of 27.8 μM (∼4 mg/L) was then used for the simulation of the degradation kinetics of the remaining NAs. Estimated kNA values at KSNA values lower and slightly higher (up to 100 μM; ∼15 mg/L) than the above-mentioned KSNA value were similar (Figure S2A, Supporting Information). On the other hand, the estimated kNA and the χ2 values increased when KSNA values above 100 μM were used (Figure S2, Supporting Information). As a result, a single KSNA value of 27.8 μM was used in all simulations. The estimated kNA values ranged between 0.022 and 2.69 μmol NA/mg VSS-d (equivalent to 0.003 and 0.39 d−1 when the respective NA MW was taken into account) (Table 2). The highest kNA value was for OC, whereas 1M1-CCA had the lowest kNA value. Although lag was observed during the biodegradation of 2-EA, the kNA value for this NA was higher than for the rest of the NAs, except that for OC. Kannel and Gan26 compiled literature data and reported NA biodegradation rate values ranging between 0.008 and 0.457 d−1 for simple NAs with Z = −2 and n = 6−12. Paslawski et al.48 investigated the biodegradation of trans-4M1-CCA by an NA enrichment culture in shake flasks and reported a biodegradation rate of 11 mg/L-day, which is about 4-fold lower than the value achieved in the present study (about 44 mg/L-day) at a comparable initial NA concentration (50 and 41 mg/L, respectively). The difference in the degradation rate between the two studies is primarily attributed to differences in biomass concentration and microbial community composition. Headley et al.38 compared the biodegradation rates of cis and trans geometric isomers of 4-methyl-1-cylcohexanecarboxylic acid, 4-methyl-1-cyclohexaneacetic acid, and 3-methyl-1-cyclohexanecarboxylic acid in Athabasca River water and found that, in all cases, the trans isomers degraded more rapidly than the
of NAs in the bioactive series was due to biotransformation. Most NAs were removed within 4 days by the enrichment culture (Figure 1). Of the seven 8-carbon NAs that were
Figure 1. Time course of NA concentrations in the NA-enriched culture series during the batch biotransformation assay: (A) OA, (B) 2-EA, (C) VA, (D) CAA, (E) 1M1-CCA, (F) 2M1-CCA, (G) 3M1CCA, and (H) 4M1-CCA. Error bars represent mean values ± one standard deviation, n = 3. Broken lines are the model fit to the experimental data (see the text).
biodegraded, OA was degraded the fastest, followed by 2M1CCA, 3M1-CCA, 4M1-CCA, 2-EA, and VA (Figure 1). A very low extent of 1M1-CCA degradation was observed in 4 days, while DDA and 5β-CA were not degraded over a period of 18
Table 2. Estimated Maximum Specific Substrate Utilization Rate (kNA) for the Aerobic Biodegradation of Select Model NAs by the NA-Enriched Culture and Biodegradation Probability Estimated by Three QSAR-Based Models aerobic biodegradation probability
a
−3
model NA
kNA (μmol NA/mg VSS-day)
χ (×10 )
BIOWIN 2
BIOWIN 6
OCHEM
OA 2-EA VA CAA 1M1-CCA 2M1-CCA 3M1-CCA 4M1-CCA DAA 5β-CA
2.69 ± 0.25a 1.64 ± 0.15 0.63 ± 0.05 0.95 ± 0.08 0.022 ± 0.005 1.45 ± 0.10 1.14 ± 0.08 1.03 ± 0.06 −b −
2.77 0.17 11.37 4.40 0.41 1.35 1.01 0.81 − −
0.969 0.969 0.833 0.837 0.477 0.837 0.837 0.837 0.615 0.007
0.890 0.760 0.760 0.738 0.761 0.565 0.565 0.565 0.308 0.032
1.000 0.620 0.550 0.850 0.460 0.590 0.580 0.650 0.340 0.070
2
Estimate ± standard error. bBiodegradation not observed. 7912
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NAs. An explanation on the effect of branching and its location on the biodegradation of acyclic and monocyclic NAs is provided in a subsequent section. Recent advancements in NA analysis have facilitated the identification of exact NA molecular structures.51−54 However, given the broad spectrum of NA molecular structures in OSPW and oil field and refinery wastewaters,4−9,20 testing the biodegradability of each NA is impossible. The capacity of the BIOWIN 2, BIOWIN 6, and OCHEM models to estimate the biodegradation probability of the 10 selected model NAs was tested, and results are shown in Table 2. As mentioned above, during the aerobic biotransformation assay, seven model NAs were removed completely, 1M1-CCA was not removed significantly, and DDA and 5β-CA were completely recalcitrant. On the basis of these experimental results, the biodegradation probability estimated by the OCHEM model was more consistent with the experimental data, as relatively low probability values (i.e., < 0.50) were assigned to the three NAs mentioned above, which either did not degrade significantly or were completely recalcitrant. All other probability scores by OCHEM were above the value of 0.460, which was assigned to 1M1-CCA. Among the three recalcitrant NAs, BIOWIN 2 and BIOWIN 6 assigned a biodegradation probability below 0.5 for only two of them, and false positives (i.e., NAs that were experimentally found to be recalcitrant are predicted to have a high biodegradation probability) are different NAs based on the BIOWIN 2 and BIOWIN 6 models. A relatively high (>0.615) biodegradation probability was estimated for DAA and 1M1-CCA by BIOWIN 2 and BIOWIN 6, respectively. Complete agreement among all three models was for 5β-CA assigning a biodegradation probability less than 0.070 (Table 2). Thus, compared to the experimental results, the estimated biodegradation probability by the OCHEM model was more consistent than the two BIOWIN models. To further test the capacity of the three QSAR-based models to estimate the biodegradation probability of NAs with variable alkyl side chain substitution and cyclicity, alkyl-substituted acyclic, monocyclic, and dicyclic NAs, as well as a tricyclic NA were considered, and results are shown in Table S3 (Supporting Information). The OCHEM and BIOWIN 6 models consistently resulted in low biodegradation probability (
