Patrick W. Folsom

8-2 Fluorotelomer alcohol aerobic soil biodegradation: Pathways, metabolites, and metabolite yields

The biodegradation pathways and metabolite yields of [3-(14)C] 8-2 fluorotelomer alcohol [8-2 FTOH, F(CF(2))(7)(14)CF(2)CH(2)CH(2)OH) in aerobic soils were investigated. Studies were conducted under closed (static) and continuous headspace air flow to assess differences in degradation rate and metabolite concentrations in soil and headspace. Aerobic degradation pathways in soils were in general similar to those in aerobic sludge and bacterial culture. (14)C mass balance was achieved in soils incubated for up to 7 months. Up to 35% (14)C dosed was irreversibly bound to soils and was only recoverable by soil combustion. The average PFOA yield was approximately 25%. Perfluorohexanoic acid (PFHxA) yield reached approximately 4%. (14)CO(2) yield was 6.8% under continuous air flow for 33 days. Three metabolites not previously identified in environmental samples were detected: 3-OH-7-3 acid [F(CF(2))(7)CHOHCH(2)COOH], 7-2 FT ketone [F(CF(2))(7)COCH(3)] and 2H-PFOA [F(CF(2))(6)CFHCOOH]. No perfluorononanoic acid (PFNA) was observed. The formation of 2H-PFOA, PFHxA, and (14)CO(2) shows that multiple -CF(2)- groups were removed from 8-2 FTOH. 7-3 Acid [F(CF(2))(7)CH(2)CH(2)COOH] reached a yield of 11% at day 7 and did not change thereafter. 7-3 Acid was incubated in aerobic soil and did not degrade to PFOA. 7-2 sFTOH [F(CF(2))(7)CH(OH)CH(3)], a transient metabolite, was incubated and degraded principally to PFOA. 7-3 Acid may be a unique metabolite from 8-2 FTOH biodegradation. The terminal ratio of PFOA to 7-3 acid ranged between 1.8-2.5 in soils and 0.6-3.2 in activated sludge, sediment, and mixed bacterial culture. This ratio may be useful in evaluating environmental samples to distinguish the potential contribution of 8-2 FTOH biodegradation to PFOA observed versus PFOA originating from other sources.

6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment plants

The aerobic biotransformation of 6:2 FTS salt [F(CF2)6CH2CH2SO3- K+] was determined in closed bottles for 90d in diluted activated sludge from three waste water treatment plants (WWTPs) to compare its biotransformation potential with that of 6:2 FTOH [F(CF2)6CH2CH2OH]. The 6:2 FTS biotransformation was relatively slow, with 63.7% remaining at day 90 and all observed transformation products together accounting for 6.3% of the initial 6:2 FTS applied. The overall mass balance (6:2 FTS plus observed transformation products) at day 90 in live and sterile treatments averaged 70% and 94%, respectively. At day 90, the stable transformation products observed were 5:3 acid [F(CF2)5CH2CH2COOH, 0.12%], PFBA [F(CF2)3COOH, 0.14%], PFPeA [F(CF2)4COOH, 1.5%], and PFHxA [F(CF2)5COOH 1.1%]. In addition, 5:2 ketone [F(CF2)5C(O)CH3] and 5:2 sFTOH [F(CF2)5CH(OH)CH3] together accounted for 3.4% at day 90. The yield of all the stable transformation products noted above (2.9%) was 19 times lower than that of 6:2 FTOH in aerobic soil. Thus 6:2 FTS is not likely to be a major source of PFCAs and polyfluorinated acids in WWTPs. 6:2 FTOH, 6:2 FTA [F(CF2)6CH2COOH], and PFHpA [F(CF2)6COOH] were not observed during the 90-d incubation. 6:2 FTS primary biotransformation bypassed 6:2 FTOH to form 6:2 FTUA [F(CF2)5CF=CHCOOH], which was subsequently degraded via pathways similar to 6:2 FTOH biotransformation. A substantial fraction of initially dosed 6:2 FTS (24%) may be irreversibly bound to diluted activated sludge catalyzed by microbial enzymes. The relatively slow 6:2 FTS degradation in activated sludge may be due to microbial aerobic de-sulfonation of 6:2 FTS, required for 6:2 FTS further biotransformation, being a rate-limiting step in microorganisms of activated sludge in WWTPs.

6-2 Fluorotelomer alcohol aerobic biodegradation in soil and mixed bacterial culture

The first studies to explore 6-2 fluorotelomer alcohol [6-2 FTOH, F(CF(2))(6)CH(2)CH(2)OH] aerobic biodegradation are described. Biodegradation yields and metabolite concentrations were determined in mixed bacterial culture (90d) and aerobic soil (180d). 6-2 FTOH primary degradation half-life was less than 2d in both. The overall mass balance in mixed bacterial culture (day 90) was approximately 60%. At day 90, the molar yield was 6% for 6-2 FTA [F(CF(2))(6)CH(2)COOH], 23% for 6-2 FTUA [F(CF(2))(5)CFCHCOOH], 16% for 5-2 sFTOH [F(CF(2))(5)CHOHCH(3)], 6% for 5-3 acid [F(CF(2))(5)CH(2)CH(2)COOH], and 5% for PFHxA [F(CF(2))(5)COOH]. The overall mass balance in aerobic soil was approximately 67% (day 180). At day 180, the major terminal metabolites were PFPeA, [F(CF(2))(4)COOH, 30%], PFHxA (8%), PFBA [F(CF(2))(3)COOH, 2%], and 5-3 acid (15%). A new metabolite 4-3 acid [F(CF(2))(4)CH(2)CH(2)COOH] accounted for 1%, 6-2 FTOH for 3%, and 5-2 sFTOH for 7%. Based on 8-2 FTOH aerobic biodegradation pathways, PFHxA was expected in greatest yield from 6-2 FTOH degradation. However, PFPeA was observed in greatest yield in soil, suggesting a preference for alternate degradation pathways. Selected metabolites were also studied in aerobic soil. 5-3 Acid degraded to only 4-3 acid with a molar yield of 2.3%. 5-2 sFTOH degraded to PFPeA and PFHxA, and 5-2 FT Ketone [F(CF(2))(5)COCH(3)] degraded to 5-2 sFTOH, suggesting that 5-2 sFTOH is the direct precursor to PFPeA and PFHxA. Another new metabolite, 5-3 ketone aldehyde [F(CF(2))(5)COCH(2)CHO] was also identified in mixed bacterial culture. The formation of PFBA, PFPeA, and 4-3 acid indicates that multiple -CF(2)- groups in 6-2 FTOH were removed during microbial biodegradation.

Aerobic biodegradation of [14C] 6:2 fluorotelomer alcohol in a flow-through soil incubation system.

The aerobic biodegradation of [1,2-(14)C] 6:2 FTOH [F(CF(2))(6)(14)CH(2)(14)CH(2)OH] in a flow-through soil incubation system is described. Soil samples dosed with [1,2-(14)C] 6:2 FTOH were analyzed by liquid scintillation counting, LC/ARC (liquid chromatography/accurate radioisotope counting), LC/MS/MS, and thermal combustion to account for 6:2 FTOH and its transformation products over 84 d. Half of the [1,2-(14)C] 6:2 FTOH disappeared from soil in 1.3 d, undergoing simultaneous microbial degradation and partitioning of volatile transformation product(s) and the 6:2 FTOH precursor into the air phase. The overall (14)C (radioactivity) mass balance in live and sterile treatments was 77-87% over 84-d incubation. In the live test system, 36% of total (14)C dosed was captured in the airflow (headspace), 25% as soil-bound residues recovered via thermal combustion, and 16% as soil extractable. After 84 d, [(14)C] 5:2 sFTOH [F(CF(2))(5)CH(OH)(14)CH(3)] was the dominant transformation product with 16% molar yield and primarily detected in the airflow. The airflow also contained [1,2-(14)C] 6:2 FTOH and (14)CO(2) at 14% and 6% of total (14)C dosed, respectively. The other significant stable transformation products, all detected in soil, were 5:3 acid [F(CF(2))(5)CH(2)CH(2)COOH, 12%], PFHxA [F(CF(2))(5)COOH, 4.5%] and PFPeA [F(CF(2))(4)COOH, 4.2%]. Soil-bound residues as well as conjugates between fluorinated transformation products and dissolved soil components were only observed in the live test system and absent in the sterile soil, suggesting that such binding and complexation are microbially or enzymatically driven processes. At day 84, 5:3 acid is postulated to be the major transformation product in soil-bound residues, which may not be available for further biodegradation in soil environment.

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

Abstract A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C3–C6 dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746.

6:2 Fluorotelomer alcohol biotransformation in an aerobic river sediment system

The 6:2 FTOH [F(CF(2))(6)CH(2)CH(2)OH] is a major raw material being used to replace 8:2 FTOH [F(CF(2))(8)CH(2)CH(2)OH] to make FTOH-based products for industrial and consumer applications. A novel aerobic sediment experimental system containing 20 g wet sediment and 30 mL aqueous solution was developed to study 6:2 FTOH biotransformation in river sediment. 6:2 FTOH was dosed into the sediment to follow its biotransformation and to analyze transformation products over 100 d. The primary 6:2 FTOH biotransformation in the aerobic sediment system was rapid (T(1/2)<2d). 5:3 acid [F(CF(2))(5)CH(2)CH(2)COOH] was observed as the predominant polyfluorinated acid on day 100 (22.4 mol%), higher than the sum of perfluoropentanoic acid (10.4 mol%), perfluorohexanoic acid (8.4 mol%), and perfluorobutanoic acid (1.5 mol%). Perfluoroheptanoic acid was not observed during 6:2 FTOH biotransformation. The 5:3 acid can be further degraded to 4:3 acid [F(CF(2))(4)CH(2)CH(2)COOH, 2.7 mol%]. This suggests that microbes in the river sediment selectively degraded 6:2 FTOH more toward 5:3 and 4:3 acids compared with soil. Most of the observed 5:3 acid formed bound residues with sediment organic components and can only be quantitatively recovered by post-treatment with NaOH and ENVI-Carb™ carbon. The 6:2 FTCA [F(CF(2))(6)CH(2)COOH], 6:2 FTUCA [F(CF(2))(5)CF=CHCOOH], 5:2 ketone [F(CF(2))(5)C(O)CH(3)], and 5:2 sFTOH [F(CF(2))(5)CH(OH)CH(3)] were major transient intermediates during 6:2 FTOH biotransformation in the sediment system. These results suggest that if 6:2 FTOH or 6:2 FTOH-based materials were released to the river or marine sediment, poly- and per-fluorinated carboxylates could be produced.

Chemoenzymatic production of 1,5-dimethyl-2-piperidone

Abstract A chemoenzymatic process for the preparation of 1,5-dimethyl-2-piperidone (1,5-DMPD) from 2-methylglutaronitrile (MGN) has been demonstrated. MGN was first hydrolyzed to 4-cyanopentanoic acid (4-CPA) ammonium salt using the nitrilase activity of immobilized Acidovorax facilis 72W cells. The hydrolysis reaction produced 4-CPA ammonium salt with greater than 98% regioselectivity at 100% conversion, and at concentrations of 170–210 g 4-CPA/l. Catalyst productivities of at least 1000 g 4-CPA/g dry cell weight (dcw) of immobilized cells were achieved by recycling the immobilized-cell catalyst in consecutive stirred-batch reactions. After recovery of the immobilized cell catalyst for reuse, the 4-CPA ammonium salt in the aqueous product mixture was directly converted to 1,5-DMPD by low-pressure catalytic hydrogenation in the presence of added methylamine.

6:2 Fluorotelomer alcohol aerobic biotransformation in activated sludge from two domestic wastewater treatment plants.

6:2 Fluorotelomer alcohol [6:2 FTOH, F(CF2)6CH2CH2OH] is a major basic chemical being used to manufacture FTOH-based products. After the end of use, 6:2 FTOH-based products may be released to domestic wastewater treatment plants (WWTPs) as a first major environmental entry point. Activated sludge collected from two WWTPs was dosed with 6:2 FTOH to investigate its biotransformation rate and to identify major transformation products. The volatile 5:2 sFTOH [F(CF2)5CH(OH)CH3] is the most abundant transformation product and accounted for an average of 40mol% of initially dosed 6:2 FTOH after two months of incubation with activated sludge, with 30mol% detected in the headspace. PFPeA [F(CF2)4COOH] averaged 4.4mol% after two months, 2.4-7 times lower than that in sediment and soils. The much lower level of PFPeA formed in activated sludge compared with soil indicates that microbial populations in activated sludge may lack enzymes or suitable environment conditions to promote rapid 5:2 sFTOH decarboxylation to form PFPeA, resulting in more 5:2 sFTOH partitioned to the headspace. PFHxA [F(CF2)5COOH] and 5:3 [F(CF2)5CH2CH2COOH] acid are major non-volatile transformation products in activated sludge. For example, PFHxA averaged 11mol% after two months, which is about 30% higher compared with sediment and soils, suggesting that microbes in WWTPs may utilize similar pathways as that in sediment and soils to convert 5:2 sFTOH to PFHxA. 5:3 Acid averaged 14mol% after two months, comparable to that in soils and slightly lower than in sediment, further confirming that 5:3 acid is a unique product of 6:2 FTOH biotransformation in the environment.

Aerobic Soil Biotransformation of 6:2 Fluorotelomer Iodide

6:2 FTI [F(CF2)6CH2CH2I] is a principal industrial raw material used to manufacture 6:2 FTOH [F(CF2)6CH2CH2OH] and 6:2 FTOH-based products and could enter aerobic environments from possible industrial emissions where it is manufactured. This is the first study to assess 6:2 FTI aerobic soil biotransformation, quantify transformation products, and elucidate its biotransformation pathways. 6:2 FTI biotransformation led to 6:2 FTOH as a key intermediate, which was subsequently biotransformed to other significant transformation products, including PFPeA [F(CF2)4COOH, 20 mol % at day 91], 5:3 acid [F(CF2)5CH2CH2COOH, 16 mol %], PFHxA [F(CF2)5COOH, 3.8 mol %], and 4:3 acid [F(CF2)4CH2CH2COOH, 3.0 mol %]. 6:2 FTI biotransformation also led to a significant level of PFHpA [F(CF2)6COOH, 16 mol % at day 91], perhaps via another putative intermediate, 6:2 FTUI [F(CF2)6CH ═ CHI], whose molecular identity and further biotransformation were not verified because of the lack of an authentic standard. Total recovery of the aforementioned per- and polyfluorocarboxylates accounted for 59 mol % of initially applied 6:2 FTI by day 91, in comparison to 56 mol % when soil was dosed with 6:2 FTOH, which did not lead to PFHpA. Thus, were 6:2 FTI to be released from its manufacture and undergo soil microbial biotransformation, it could form PFPeA, PFHpA, PFHxA, 5:3 acid, and 4:3 acid in the environment.