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Biotransformation of Nitrosamines and Precursor Secondary Amines under Methanogenic Conditions Ulas Tezel,† Lokesh P. Padhye,†,‡ Ching-Hua Huang,†,* and Spyros G. Pavlostathis†,* † ‡
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States Geosyntec Consultants, Kennesaw, Georgia 30144, United States
bS Supporting Information ABSTRACT: The biotransformation potential of six nitrosamines and their precursor secondary amines by a mixed methanogenic culture was investigated. Among the six nitrosamines tested, N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), and N-nitrosopyrrolidine (NPYR) were almost completely degraded but only when degradable electron donors were available. On the contrary, N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), and N-nitrosodibutylamine (NDBA) were not degraded. Three precursor secondary amines, corresponding to the three biodegradable nitrosamines, were also completely utilized even with very low levels of available electron donors. The secondary amine precursors of the three, nonbiodegradable nitrosamines were also recalcitrant. A bioassay conducted to elucidate the biotransformation pathway of NDMA in the mixed methanogenic culture using H2 as the electron donor showed that NDMA was utilized as an electron acceptor and transformed to dimethylamine (DMA), which in turn was degraded to ammonia and methane. The H2 threshold concentration for NDMA bioreduction ranged between 0.0017 and 0.031 atm. Such a high H2 threshold concentration suggests that in mixed methanogenic cultures, NDMA reducers are weak competitors to other, H2-consuming microbial species, such as homoacetogens and methanogens. Thus, complete removal of nitrosamines in anaerobic digestion systems, where the H2 partial pressure is typically below 104 atm, is difficult to achieve.
’ INTRODUCTION Nitrosamines (NISA) are an emerging group of disinfection byproducts and suspected carcinogens.1 Among all nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPYR), N-nitrosodiethylamine (NDEA), N-nitrosodi-N-propylamine (NDPA), and N-nitrosodi-N-butylamine (NDBA), are of particular interest because they are listed in the Unregulated Contaminant Monitoring Regulation (UCMR 2)2 as well as the recently proposed Contaminant Candidate List 3 (CCL 3) by the U.S. Environmental Protection Agency.3 Secondary amines (SAN) are well-known nitrosamine precursors that can yield corresponding nitrosamines via Nnitrosation,4 during disinfection,1,5,6 or at different stages of water and wastewater treatment.7,8 Recent studies have reported the presence of nitrosamines and their precursors in both the influent and effluent of municipal wastewater treatment plants throughout the U.S. and Canada.7,9 Aerobic degradation and mineralization of nitrosamines, particularly NDMA, has been observed in mixed microbial cultures.10,11 Biodegradation of nitrosamines has also been reported for indigenous soil bacteria under aerobic conditions.12,13 A recent study showed that aerobic NDMA biotransformation was cometabolic, achieved by various monooxygenases, such as soluble methane monooxygenase, propane monooxygenase and toluene-4-monooxygenase.14,15 Aerobic, cometabolic NDMA biotransformation takes place either through the cleavage of the N—N bond r 2011 American Chemical Society
(denitrosation)14 or by α-hydroxylation of the methyl group followed by a demethylation.16 NDMA biodegradation under anaerobic conditions has also been reported for indigenous soil microorganisms and biofilm reactors; however, the rate and extent of biodegradation under anaerobic conditions were significantly lower than under aerobic conditions.17,18 Biotransformation of secondary amines via demethylation has also been reported under both aerobic and anaerobic conditions.19 Methanogens are also known to utilize secondary amines, such as DMA, and convert them to methane.20,21 Recently, Padhye et al.9 showed that nitrosamines, particularly NDMA, are abundant in municipal primary and waste activated sludge at concentrations up to 10-fold higher than those found in municipal wastewater. Considering that many of the NISA are hydrophilic and have a low affinity to sorb on sludge solids, high NISA concentrations found in municipal sludge were therefore attributed to the in situ formation of NISA through nitrosation of nitrogen-bearing, sludge-associated organic compounds, such as quaternary ammonium compounds and cationic polymers.9,22,23 Moreover, it was shown that NISA present in municipal sludge were partially removed during anaerobic digestion.9 The study Received: February 17, 2011 Accepted: August 24, 2011 Revised: August 16, 2011 Published: August 24, 2011 8290
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Environmental Science & Technology concluded that bioavailable electron donors are necessary for the removal of NISA under methanogenic conditions, but other factors contributing to the biological NISA removal were not explored, neither was the biotransformation pathway elucidated.9 Considering that anaerobic digester supernatant and/or liquid stream resulting from dewatering processes after sludge digestion are typically returned to the head of the wastewater treatment plant, effective removal of NISA during anaerobic sludge digestion is desirable. The objectives of this study were to (a) assess the biotransformation potential of the above-mentioned six nitrosamines and their corresponding secondary amine precursors [dimethylamine (DMA), methylethylamine (MEA), pyrrolidine (PYR), diethylamine (DEA), di-N-propylamine (DPA), and di-N-butylamine (DBA)] by a mixed methanogenic culture; and (b) elucidate the NDMA biodegradation pathway under methanogenic conditions.
’ MATERIALS AND METHODS Chemicals. All NISA and SAN compounds used in this study were obtained at >98% purity from Sigma-Aldrich (St. Louis, MO). The isotopes NDMA-d6, NDEA-d10, NDPA-d14, and DMA-d6 hydrochloride were obtained from Cambridge Isotope Laboratories (Andover, MA). Molecular structure, formula, weight, and measured logP values of each compound are provided in the Supporting Information (Tables S1 and S2). The NISA and corresponding SAN are grouped according to their logP values. Total electron density of NDMA, 1,1-dimethylhydrazine (UDMH), a potential intermediate of NDMA reduction, and DMA was mapped at electrostatic potential values between 0.01 and 0.01 e/au3 and an isovalue of 0.0004 e/au3 after geometry optimization and energy minimization using 6-31G level HarteFock theory and CPCM solvency model performed with Gaussian 03 (Gaussian, Inc.; Wallingford, CT). Batch NISA and SAN Biotransformation Assays. A batch assay was performed to investigate the biotransformation potential of six nitrosamines: NDMA, NDEA, NDPA, NDBA, NMEA, and NPYR. The assay was conducted in 160-mL serum bottles (125 mL liquid volume) sealed with rubber stoppers and aluminum crimps and flushed with helium gas for 15 min before any liquid addition. The mixed methanogenic culture used in this study has been previously described.24 A sample of 80 mL of the mixed methanogenic culture taken at the end of a 7-day feeding cycle was anaerobically transferred to each serum bottle along with 40 mL of culture media, which was buffered with 0.9 g/L K2HPO4, 0.5 g/L KH2PO4, and 3.5 g/L NaHCO3 (pH 7.5). Dextrin and peptone (DP) solution, used as the carbon and energy source, and a sample of composite nitrosamines solution were added. The total liquid volume was then brought to 125 mL with deionized (DI) water. The initial biomass and DP concentrations in the bottles were 1.7 g VS/L and 1200 mg COD/L, respectively. The initial concentration of each NISA in the bottles was 16 μM. One additional culture series, which consisted of only inoculum, culture media, DP and DI water was prepared and used as a reference. An abiotic media control was prepared identically to the culture series, except that culture was not added. All cultures, the reference, and the abiotic media controls were prepared in triplicate. All bottles were kept in the dark at 35 °C and agitated by hand daily. After a significant decrease in the concentration of NDMA, NMEA, and NPYR in the active, nitrosamine-amended cultures (see below), these cultures were reamended with a mixture of
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these three nitrosamines (second feeding cycle). The cultures were supplemented with DP at the same level as at the beginning of the assay (see above). The second cycle was followed by another cycle that was similar to the previous one in terms of nitrosamine amendment, but the cultures were not supplemented with any external energy and carbon source at the beginning of this cycle (third feeding cycle). In order to test the biotransformation potential of six SAN, a similar set of bottles was prepared as described above using a composite sample of SAN, with each secondary amine tested at an initial concentration of 16 μM. The same three feeding cycles, described above, were followed, except depleted SAN were added instead of NISA. Throughout the incubation period of every feeding cycle, the total gas volume and its methane and carbon dioxide content, as well as the total, liquid concentration of the six nitrosamines and six secondary amines were measured. Volatile fatty acids (VFAs) analysis was also conducted at the end of each feeding cycle. Batch NDMA Biotransformation Assay. Another batch assay was performed to elucidate the NDMA degradation pathway. The assay was conducted in 160-mL serum bottles (100 mL liquid volume). In order to inhibit methanogenesis, and thus recover DMA, the NDMA reduction product,18 as well as eliminate any competition for H2 consumption between NDMA reducers and hydrogenotrophic methanogens, 1 g of 2-bromoethanesulfonic acid (BES) was added to each bottle. The bottles were then sealed with rubber stoppers and aluminum crimps and flushed with helium gas for 15 min before adding any liquid. A sample of 20 mL of the mixed methanogenic culture taken at the end of a 7-day feeding cycle was anaerobically transferred to each serum bottle along with 78 mL of culture media, which was buffered with 0.9 g/L K2HPO4, 0.5 g/L KH2PO4, and 3.5 g/L NaHCO3 (pH 7.5). The bottles’ headspace was set at 1 atm with a 80:20 (v/v) mixture of H2:CO2 for H2 to be used as the electron donor for the reduction of NDMA. NDMA was added to all bottles at an initial concentration of 16 μM. Another four sets of bottles were prepared as follows: the first set contained culture, culture media and NDMA; the second set contained culture, culture media, BES and H2+CO2; the third set contained culture, culture media, and BES; and the final set contained culture, culture media, BES and 16 μM DMA. Throughout the incubation period, the headspace gas pressure and its methane, carbon dioxide and hydrogen content, as well as the total, liquid concentration of NDMA and DMA were measured. VFAs analysis was also conducted at the end of the incubation period. Analytical Methods. Total solids (TS), volatile solids (VS), chemical oxygen demand (COD), and pH were measured according to Standard Methods.25 Total gas production, headspace gas pressure, gas composition (i.e., CH4, H2, CO2) and VFAs (i.e., C2C7) were measured as previously described.24,26 Analysis of NISA and SAN was performed as follows: two, 2mL samples were removed from the culture bottles. One sample, used for nitrosamine analysis, was amended with 500 μg/L each of three surrogate standards, NDMA-d6, NDEA-d10, and NDPAd14. The second sample, used for secondary amine analysis, was adjusted to pH 9.0 with a 10 mM borate buffer and amended with 100 μg/L DMA-d6 as a surrogate standard. The samples were first vortexed, then centrifuged at 12 000 rpm for 20 min, and finally filtered through 0.22 μm nylon membrane filters (Pall Life Sciences, East Hills, NY). The first set of filtrates was extracted with 2 mL of dichloromethane and the extracts were analyzed for nitrosamines. The second set of filtrates was amended with a 8291
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Figure 1. (A) Total gas, methane and carbon dioxide production, and (B) NDMA, NMEA and NPYR consumption by a mixed methanogenic culture in the course of three feeding cycles with NISA amendments. In the first two cycles, the culture was supplied with DP, whereas external carbon and energy source was not provided with the last NISA amendment (third cycle) (Error bars represent one standard deviation of the means; n = 3).
5 mM of 4-methoxybenzenesulfonyl chloride (MBSC) solution, agitated for 2 h, extracted with 2 mL of dichloromethane for 2 min, and the extracts were then analyzed for derivatized secondary amines. Quantification of nitrosamines and derivatized secondary amines was performed by GC/MS as previously described.9
’ RESULTS AND DISCUSSION Biotransformation of NISA. The first cycle of this assay, testing the biotransformation potential of the six nitrosamines in a mixed methanogenic culture, lasted 75 days. The total volume of the gas produced in nitrosamine-amended and the reference cultures at the end of the first incubation period was 97 ( 1 and 103 ( 1 mL, respectively (Figure 1A and Supporting Information Figure S1). The methane content of the gas produced in the nitrosamine-amended cultures and reference was 66.7 ( 0.3 and 64.3 ( 0.4%, respectively (Figure 1A and Supporting Information Figure S1). Thus, the six nitrosamines did not inhibit the microbial activity of the mixed methanogenic culture at the applied concentration, which is higher than that typically found in municipal anaerobic digesters.7 Similarly, reamendment of the same cultures with three NISA did not affect microbial activity over an incubation period of 96 days (second feeding cycle) (Figure 1A and Supporting Information Figure S1). As expected, during the third feeding cycle, which lasted for 105 days, the fermentative/methanogenic activity of the cultures was very low and related to endogenous decay because an external carbon and energy source was not added for this cycle (Figure 1A and Supporting Information Figure S1). To verify that abiotic processes were not responsible for the loss of nitrosamines in the above-described experiments, media controls without any biomass were also monitored at the same
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time intervals as those for the active culture series. The change in nitrosamine concentration in the controls was less than 5% of the initial concentration for all six nitrosamines over the 276-days incubation period (Supporting Information Figure S2A). During incubation of the NISA-amended active cultures, samples were collected and analyzed at 0, 10, 31, and 52 days for cycle 1 (Figure 1B). Over time, there was a significant decrease in the concentration of Group-1 nitrosamines (Supporting Information Table S1), which have relatively low logP values, that is., NDMA, NMEA, and NPYR. At 52 days, about 25% of NDMA and 7% of NPYR remained, and NMEA was not detected (minimum detection limit about 0.28 μM for the GC/MS method used). In contrast, the concentration of the Group-2 nitrosamines (Supporting Information Table S1), which have relatively high logP values, that is, NDEA, NDPA, and NDBA, did not show any significant change (Supporting Information Figure S3). Given that the Group-1 nitrosamines have negligible adsorption to biomass, the decrease in the concentration of the Group-1 nitrosamines over the incubation time was attributed to biotransformation. Because the concentration of the Group-1 nitrosamines approached the minimum detection limits at the end of 52-days incubation, the cultures were reamended with 16 μM of NDMA, NMEA, and NPYR on day 75 along with DP (beginning of cycle 2) (Figure 1B). Nitrosamine analysis at the end of this cycle showed that 21% of NDMA remained, whereas NMEA and NPYR were completely depleted. The trend of NISA concentrations was similar to that observed during the first feeding cycle. In the third feeding cycle, however, the rate of degradation of these three nitrosamines initially amended at 19.4, 22.1, and 16.2 μM, respectively, was noticeably lower. About 2% of NMEA and 15% of NPYR remained after 105 days of incubation, but only 50% of the initial NDMA was consumed (Figure 1B). VFAs were not detected at the end of any feeding cycle. The results of this assay show that nitrosamines can be degraded under fermentative/methanogenic conditions, but the rate of nitrosamine degradation is affected by the availability of degradable electron donor(s). Furthermore, adsorption to biomass and molecular structure may also affect the biodegradation of nitrosamines. Nitrosamines with relatively high logP values, containing symmetric, long-chain alkyl groups were more resistant to biodegradation than nitrosamines with relatively low logP values and shorter or asymmetrical alkyl chains. As a result, NMEA had the highest rate of biotransformation, followed by NPYR and then NDMA. On the contrary, NDEA, NDPA and NDBA were recalcitrant under the conditions used in this study. Similar results were obtained in our previous study performed with different anaerobic digester microcosms.9 Although anaerobic degradation of NISA, particularly NDMA, has been demonstrated in other studies,13,18,27 the factors affecting the biodegradation of NISA as demonstrated in this study adds to our understanding of their fate and the development of strategies for the removal of NISA in anaerobic biological systems. Biotransformation of SAN. The first cycle of this assay testing the biotransformation potential of six secondary amines in a mixed methanogenic culture lasted 75 days. The total volume of gas produced in the secondary amines-amended cultures and the reference at the end of the incubation period was 98 ( 1 and 103 ( 1 mL, respectively (Figure 2A and Supporting Information Figure S1). The methane content of the gas produced in the secondary amines-amended cultures and the reference was 67.3 ( 0.7 and 64.3 ( 0.4%, respectively (Figure 2A and Supporting Information Figure S1). Similarly, reamendment of 8292
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Figure 2. (A) Total gas, methane and carbon dioxide production, and (B) DMA, MEA and PYR consumption by a mixed methanogenic culture in the course of three feeding cycles with SAN amendments. In the first two cycles, the culture was supplied with DP, whereas external carbon and energy source was not provided with the last SAN amendment (third cycle) (Error bars represent one standard deviation of the means; n = 3).
the same cultures with the secondary amines did not affect the microbial activity over an incubation period of 96 days (cycle 2) (Figure 2A and Supporting Information Figure S1). Thus, the six secondary amines tested in this study did not inhibit the microbial activity of the mixed, methanogenic culture at the applied concentration. During the third feeding cycle, which lasted for 105 days, the fermentative/methanogenic activity of the cultures was very low as discussed above (Figure 2A and Supporting Information Figure S1). The SAN concentrations in the abiotic controls decreased to some degree initially, but remained constant for the remainder of the 276-days incubation (Supporting Information Figure S2B). Similar to the nitrosamine results, on day 52 the Group-1 secondary amines (Supporting Information Table S2) which have relatively low logP values, that is, DMA, MEA, and PYR, had a much lower concentration than initially. About 15 and 35% of DMA and MEA, respectively, remained, whereas PYR was not detected (minimum detection limit about 0.14 μM for the GC/MS method used). The Group-2 secondary amines (Supporting Information Table S2) which have relatively high logP values, that is, DEA, DPA, and DBA, were not degraded significantly in the first 30 days (cycle 1) (Supporting Information Figure S4). At the end of the first feeding cycle, the culture was reamended with DMA, MEA, and PYR for the two subsequent feeding cycles as was described above. All three secondary amines were depleted by the end of both cycles (cycles 2 and 3) (Figure 2B). The concentration of the Group-2 secondary amines did not change significantly over the 276-days incubation period, except for DEA, for which about 39% remained (Supporting Information Figure S4). However, given the fact that the DEA concentration also declined in the abiotic media controls to some extent (Supporting Information Figure S2B), biotransformation of
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this secondary amine by the mixed methanogenic culture is not conclusive. VFAs were not detected at the end of any feeding cycle. The results of this assay show that Group-1 secondary amines can be rapidly degraded under fermentative/methanogenic conditions even when relatively low levels of degradable electron donor(s) are available. On the contrary, similar to nitrosamines, secondary amines with relatively high logP values and symmetric, long-chain alkyl groups were recalcitrant under the conditions of the present study. Thus, as is the case for nitrosamines, hydrophobicity and molecular structure play a significant role on the biotransformation of secondary amines. Chung et al.18 studied the biodegradation of NDMA in a hydrogen-based membrane biofilm bioreactor and suggested that NDMA serves as an electron acceptor and is biologically reduced to its corresponding secondary amine, DMA, while H2 serves as the electron donor. Although the proposed NDMA pathway was not confirmed in the previous study, others who have investigated the abiotic reduction of NDMA by metals have confirmed that such a reduction pathway is possible.28,29 Given the observation that three nitrosamines were degraded (possibly to their corresponding secondary amines), while the others did not, free energy considerations may be informative. Gibb’s free energy values for the reduction of the six nitrosamines to their corresponding secondary amines (i.e., denitrosation/nitroso reduction) using H2 as the electron donor were calculated using a semiempirical molecular model. The reduction of all six NISA is exergonic, with free energy values varying from 234.5 to 253.3 kJ/mol NISA for NDMA, NMEA, and NPYR, and from 261.3 to 268 kJ/mol NISA for NDEA, NDPA, and NDBA (Supporting Information Table S3). It is noteworthy that the first set of three NISA, which were transformed by the mixed methanogenic culture in this study, has a lower free energy yield than the second set of three NISA, which were recalcitrant under the conditions of the present study (Supporting Information Table S3). Therefore, nitrosamine reduction is most likely not controlled by energetics. The recalcitrance of Group-2 secondary amines under highly reduced conditions may indicate that the secondary amine moiety contributes to the recalcitrance of the corresponding nitrosamines, possibly because of hydrophobicity and/or steric effect as discussed below. Chung et al.18 showed that the H2 concentration is a limiting factor for NDMA bioreduction. In their biofilm system, NDMA bioreduction was outcompeted by nitrate and sulfate reduction processes, indicating that the H2 threshold for NDMA reduction is higher than that of nitrate and sulfate reduction processes. In a mixed methanogenic community, like the one used in the present study, H2 is produced during the fermentation of complex organics, long-chain and volatile fatty acids, whereas it is consumed during acetogenesis, homoacetogenesis and methanogenesis (Supporting Information Figure S5). In the present study, nitrosamine degradation was only carried out when electron donors were available and active culture conditions were maintained, while degradation ceased as soon as the electron donors were completely transformed to methane. Thus, nitrosamine reduction may have to compete with other, H2-consuming processes, including reduction of alternative, terminal electron acceptors, if present. Therefore, H2 threshold levels may control the rate and extent of nitrosamine bioreduction as discussed below. NDMA Biotransformation Pathway. A batch assay was setup to elucidate the NDMA biotransformation pathway in a mixed 8293
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Table 1. Energetics of Different Electron Accepting Processes Using H2 as the Electron Donor H2 threshold electron acceptor
reaction
Gibb’s free energy (ΔG00 ) kJ/mol H2a
redox potential (E00 ) V
atm ( 106)b
nMc
O2
H2 + 1/2 O2 T H2O
237.2
+0.82
