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LETTERS TO NATURE allowing for the van der Waals radii of the attached hydrogens, the `free space' dimension of the channel in this direction (left to right in Fig. 2A) is close to 20 A. The distance between a Cu(n) of one pair and the nearest Cu(ii) of another pair across a channel is 15.64(2) A. We suggest that interpenetration of the sort observed for solvated Cu(tcp)CuBF4 does not occur with solvated Cu(tpp)CuBF4 because the required close approach of the Cu(i) centres from separate frameworks is precluded by the four bulky pyridyl units around each metal ; by contrast it would be difficult to find four less sterically demanding donors than the nitrile ligands surrounding the Cu( t) in Cu(tcp)CuBF4. Paired metalloporphyrins have been studied as models for the `special pair' of the photosynthetic system'; paired frameworks related to that described here with a range of metallo-tcp units in place of Cu(tcp) could perhaps be made, and may furnish interesting photophysical and photochemical behaviour. With regard to future studies, porphyrins and phthalocyanins are particularly alluring as building blocks for networks with potential applications as microporous heterogeneous catalysts because (1) their relative rigidity and large size, in the correct circumstances, could be used to generate correspondingly large channels and cavities, (2) they show a high degree of thermal stability and could therefore be put to use at elevated tempera- tures, (3) they readily incorporate a wide range of metal centres and (4) as discrete molecular species, they are known to catalyse and promote diverse processess'. The porphyrin-containing net- works described here are so delicate that they do not survive removal of nitrobenzene, so, at this early stage, materials of this general type fall far short of rivalling zeolites. We are presently exploring the possibility of putting to use the stability-enhancing chelate effect to provide more robust solids. The exercise described here was conducted to demonstrate, in the simplest way we could visualize, what might be possible with porphyrin building blocks. The fact that the PtS network did assemble itself as intended is very encouraging with regard to future framework construction. On the basis of the results presented here, the deliberate construction of microporous heterogeneous catalysts (in which porphyrins and similar sys- tems play the dual role of active site and framework component of sufficiently large size to produce large access channels) seems a realistic prospect. The basic approach is obviously amenable to wide variation; it seems feasible that appropriately designed building blocks may afford many nets other than PtS and diamond. With regard to the prospects for catalytic activity off- ered by framework solids in general, it is encouraging that a two-dimensional Cd(n)(4,4'-bipyridine), sheet polymer contain- ing corral-like square cavities surrounded by 4,4'-bipyridine `fences'' has recently been shown to catalyse the cyanosilylation of aldehydes24. u Received 29 December 1993; accepted 5 May 1994. 1. Bein, T. (ed.) Supramolecular Architecture 88-253 (ACS Symp. Ser. No. 499, Am. Chem. Soc., Washington DC, 1992). 2. Desiraju, G. R. Crystal Engineering: The Design of Organic Solids (Elsevier, Amsterdam, 1989). 3. Fagan, P. J. & Ward, M. D. Scient. Am. 267, 28-34 (1992). 4. Simard, M., Su, D. & Wuest, J. D. J. Am. chem. Soc. 113, 4696-4698 (1991). 5. Ermer, 0. J. Am. chem. Soc. 110, 3747-3754 (1988). 6. MacNicol, D. D., McKendrick, J. J. & Wilson, D. R. Chem. Soc. Rev. 7, 65-87 (1978). 7. MacNicol, D. D. Inclusion Compounds Vol. 2, 1-45 (Academic, London, 1984). 8. Derouane, E. G., Lemos, F., Naccache, C. & Ribeiro, F. R. (eds) Zeolite Microporous Solids: Synthesis, Structure and Reactivity (NATO ASI Ser. C, Vol. 352, Kluwer Academic, Dordrecht, 1992). 9. Kerr, G. T. Scient. Am. 261, 82-87 (1989). 10. Estermann, M., McKusker, L. B., Baerlocher, C., Merrouche, A. & Kessler, H. Nature 352, 320-322 (1991). 11. Davis, M. E., Saldarriaga, C., Montes, C., Garces, J. & Crouder, C. Nature 331, 698-699 (1988). 12. Bein, T. (ed.) Supramolecular Architecture 256-273 (ACS Symp. Ser. No. 499, Am. Chem. Soc., Washington DC, 1992). 13. Hoskins, B. F. & Robson, R. J. Am. chem. Soc. 112, 1546-1554 (1990). 14. Gable, R. W., Hoskins, B. F. & Robson, R. J. chem. Soc., chem. Commun. 762-763 (1990). 15. Byrn, M. P. et al. J. Amer. chem. Soc. 115, 9480-9497 (1993). 16. Wohrle, D. in Phthalocyanins: Properties and Applications (eds Leznoff, C. C. & Lever, A. B. P.) 55-132 (VCH, New York, 1989). 17. Takemoto, K., Inaki, Y. & Ottenbrite, R. M. Functional Monomers and Polymers (Dekker, New York, 1987). 18. Collman, J. P. et al Proc. natn. Acad. Sci. U.S.A. 83, 4581-4585 (1986). 19. Gunter, M. J. et al. Inorg. Chem. 23, 283-300 (1984). 20. Fleischer, E. B. & Shachter, A. M. lnorg. Chem. 30, 3763-3769 (1991). 21. Abrahams, B. F., Hoskins, B. F. & Robson, R. J. Am. chem. Soc. 113, 3606-3607 (1991). 22. Sessler, J. L., Johnson, A. R., Lin, T.-Y. & Creager, S. E. J. Am. chem. Soc. 110, 3659-3661 (1988). 23. Walter, C. 1, Anderson, H. L. & Sanders, J. K. M. J. chem. Soc. chem. Commun. 458-460 (1993). 24. Fujita, M., Kwon, Y. J., Washizu, S. & Ogura, K. J. Am. chem. Soc. 116, 1151-1152 (1994). 25. Sheldrick, G. M. in Crystallographic Computing Vol. 3 (eds Sheldrick, G. M., Kruger, C. & Goddard, R.) 175-189 (Oxford Univ. Press, 1985). 26. Sheldrick, G. M. SHELXS-76, a Program for Crystal Structure Determination (Univ. Cambridge, 1976). SUPPLEMENTARY INFORMATION. Requests should be addressed to Mary Sheehan at the London editorial office of Nature. ACKNOWLEDGEMENTS. We thank the Australian Research Council and the American Chemical Society Petroleum Research Fund for support. Degradation of trifluoroacetate in oxic and anoxic sediments Pieter T. Visscher, Charles W. Culbertson & Ronald S. Oremland US Geological Survey, Mailstop 465, 345 Middlefield Road, Menlo Park, California 94025, USA THE deleterious effect of chlorofluorocarbons on stratospheric ozone has led to international cooperation to end their use1-3. The search for acceptable alternatives has focused on hydrofluorocarbons (HFCs) or hydrochlorofluorocarbons (HCFCs) which are attractive because they have relatively short atmospheric residence times. HFCs and HCFCs are attacked by tropospheric hydroxyl radicals, leading to the formation of trifluoroacetate (TFA)5. Most of the atmospheric TFA is deposited at the Earth's surface6, where it is thought to be highly resistant to bacterial attacks. Therefore, use of HCFCs and HFCs may lead to accumulation of TFA in soils, where it could prove toxic or inhibitory to plants and soil microbial communities''. Although little is known about the toxicity of TFA, monofluoroacetate, which occurs at low levels in some plants8 and which is susceptible to slow attack by aerobic soil microbes9, is known to be acutely toxicI6-13. Here we report that TFA can be rapidly degraded microbially under anoxic and oxic conditions. These results imply that significant microbial sinks exist in nature for the elimination of TFA from the environment. We also show that oxic degradation of TFA leads to the formation of fluoroform, a potential ozone-depleting compound with a much longer atmospheric lifetime than the parent compounds. We chose to work initially with anoxic sediments because of their ability to degrade CFCs14 and other halogenated methyl compounds such as methyl bromide' 5. We incubated anoxic sediments from a San Francisco Bay saltmarsh16 and from a freshwater lakel7" in the presence of 1.85 M 2-14C-TFA (Fig. 1). Production of CH4 (Fig. la) and 14CH4 (Fig. lb) in saltmarsh sediments only occurred when sulphate reduction was inhibited with molybdate l9 or by elimination of sulphate from the slurry salts. No 14CH4 was noted in the controls, which included autoclaved sediments containing molybdate (Fig. lb), thereby reinforcing our conclusion that this was a microbial rather than chemical phenomenon. However, unmanipulated freshwater sediments (SO,12- < 1 mM) also readily produced 14CH4 (Fig. lb), thereby demonstrating that TFA degradation can occur in other types of sediments. The unlabelled carboxyl group of TFA was therefore cleaved to CO2 , as occurs in acetoclastic methanogenesis2. Addition of 2-bromoethanesulphonic acid, an inhibitor of methanogenic bacteria'9l , blocked production of CH4 and 14CH4 . NATURE VOL 369 30 JUNE 1994 729 1994 Nature Publishing Group LETTERS TO NATURE Electron acceptor 02 N03 None TFA (M) 0.925 0.463 0.463 0.185 0.925 0.463 1.850 0.925 0.463 1.850 0.925 0.463 TABLE 1 Degradation of 2-14C-TFA in sediment slurries Incubation on time (d) 14CHF3 (Ci 1-1) 14C02 (Ci 27 0 (0) 0 27 0.60 (0.10) 0 15 0.50 (0.35) 0 15 2.55 (2.35) 0 15 0 0 15 0 0 12 0 0 18 0 0 05 (0.1) 18 0 3.05 (0.95) 8 0 0 15 0 0 15 0 0 14CH4 (Ci 0 0 0 0 0 0 0 0 0 72.95 (3.25) 34.95 (0.80) 21.45 (0.70) TFA conversion (%) 0 (0) 2.4 (0.3) 1.9 (1.5) 25.5 (23.7) 0 0 0 0 1 (0.2) 12.2 (3.9) 73.0 (3.3) 69.9 (1.6) 86.9 (2.8) Experiments were carried out with 20 ml of slurry in 57-ml serum bottles (except oxic incubations, which were performed in 160-ml serum bottles). Oxic samples were pre-incubated by shaking with exposure to the atmosphere for 20 h in order to remove all potential reducing agents. Mean of three replicate slurries are given (figures in parenthesis indicate standard deviation). Autoclaved slurries, incubated anoxically with sulphate or under oxic conditions, did not produce any 14C-labelled gases, ruling out chemical processes. Under these conditions, we did not observe the production of '4C-trifluoromethane, ' 4C-difluoromethane, '4C-methylfluoride or "CO2 in the headspaces of any of the slurries (not shown). We interpret these observations to mean that TFA is readily degraded under methanogenic conditions, but we were surprised that conditions that favoured sulphate-reduction did not result in formation of 14CO2 , which we would predict to occur for acetate21. We pursued the question of TFA mineralization by sulphate reduction in a subsequent experiment (see below). Because in our first experiment, the only end product was 14CH4i it implied that TFA underwent a complete defluorination to the level of acetate before methanogenesis. To test this hypothesis, we examined the liquid phase, as well as the gas phase, of a larger volume of saltmarsh slurry incubated without sulphate. Results clearly demonstrated that a sequential order of defluorination occurred in these sediments (Fig. 2). Thus, the disappearance of 2-14C-TFA was respectively followed by the transient appearances of HC-difluoroacetate (2-14C-DFA), ' 4C-monofluoroacetate (2-14C-MFA), 2-'4C-acetate, and eventually the production of 14CH4 . By the time 14CH4 production had ceased, after 7 days incubation, there no longer were detectable counts in the liquid phase. Mean conversion efficiencies of 2-14C-TFA to 14CH4 was 80% for this experiment, and 66-77% in the previous experiments (see below). We attribute the slightly lower efficiencies to binding of acetate to sediment surfaces, a process which makes a portion of it unavailable to microbial degradation22, as the cause for the less than 100% recovery of 2-14C-TFA as 14CH4 . We did not detect any alteration of the 214C-TFA in autoclaved controls after prolonged (6 weeks) incubation (data not shown). Additionally, sediments inhibited by 2-bromoethanesulphonic acid had only a slight loss of TFA (1520%) after 27 days incubation (data not shown). We conclude that this rapid defluorination is carried out by microorganisms, and the results with 2-bromoethanesulphonic acid suggest direct involvement of methanogens in the reductive defluorination of the TFA. The anaerobic microbial reductive dehalogenation of aromatic and aliphatic compounds has been well studied, but mostly centres on dechlorination and debromination reactions23. Defluorination of fluoroaromatics occurs in methanogenic enrichments, but fluorine is removed before ring cleavage and acetate formation24. This work is the first report of multiple reductive defluorinations of the methyl group of acetate. Fluorinated compounds have been employed as microbial metabolic inhibitors13"8. Monofluoroacetate, an intermediate of TFA degradation in our study, inhibited acetoclastic methanogenesis in lake sediments when applied at a concentration of .' 20 M (ref. 13). We observed an inhibitory effect of added unlabelled TFA on methanogenic activity at concentrations 1 M. After 10 days incubation of sulphate-free saltmarsh slurries, mean methane levels (moles per 20 ml slurry; +1 stan- 1.5 0 0 0.5 0 50 100 150 200 250 300 Time (h) FIG. 1 Production of CH4 (a) and 14CH4 (b) in anoxic sediment slurries. Sediments were homogenized anaerobically with either artificial bay water (for saltmarsh sediments)24 or with lake water (for fresh water)17'18 to yield final ratios of 1:3, sediment:water. Slurries (20 ml) were contained in 57-ml serum bottles sealed under 02-free N2 with black butyl crimp-seal stoppers. Samples received 2 Ci of 2-14C-TFA (Amersham Inc., Arlington Heights, Illinois); purity, 99.6%; specific activity, 54 mCi mmol- 1). Slurries were incubated in the dark at 23 C with constant rotary shaking (200 r.p.m.). Gas phases were subsampled (250 I) by syringe, and methane was analysed by flame-ionization gas chromatography33, whereas 14CH4 and 14C02 were analysed by gas chromatography in conjunction with proportional counting34. Retention times were (min): CH4 (0.8), CH3F (2.5) and CHF3 (3.5). At the end of the incubations, samples were injected with 2 ml of 6 M HCI and shaken overnight before determination of 14CO2 . Symbols: saltmarsh sediments incubated with 20 mM sulphate (*), without sulphate (), with 20 mM sulphate plus 2.5 mM molybdate (), without sulphate plus 5 mM bromoethanesulphonate (A), autoclaved with 20 mM sulphate and 2.5 mM molybdate (Ili), autoclaved with 20 mM sulphate (O); live freshwater sediments (A). Symbols represent the mean of three individual slurries and error bars display 1 standard deviation. 730 1994 Nature Publishing Group NATURE VOL 369 30 JUNE 1994 LETTERS TO NATURE dard deviation; n= 3) were; without additions (90 15); plus 0.1 M TFA (73 7), plus 1 M TFA (4.0 2.1), and plus 10 M TFA (2.4 0.5). Therefore, we hypothesized that our initial observation of no 14CO2 production under conditions favouring sulphate-reduction may have been caused by stronger inhibitory effects of TFA (or of MFA) on sulphate-reducers. This led us to perform a series of experiments in which lower concentrations of TFA (0.185-0.925 M) were applied. The results from these incubations clearly show that sulphate-reducers can oxidize the methyl carbon of TFA to CO2 , provided that the TFA concentration was lower than 0.925 M (Table 1). Conversion efficiencies were highest at the lowest concentration (0.463 M) of added TFA. We did not detect any degradation of TFA (that is, loss of TFA or formation of products) under conditions favouring nitrate respiration (Table 1), despite the fact that these slurries actively formed as much as 60% more unlabelled CO2 than any of the other electron-acceptors tested (data not shown). However, with O2 as electron acceptor, we detected a peak with a slightly shorter retention time than that of 14CO2 . We then used a 4 times longer column (7.3-m Porapak Q) which separated 14CHF3 (retention time, 5.8 min) from 14CO2 (retention time, 9.0 min), thereby confirming that the observed product was only "CHF3 . We only observed production of 14CHF3 when the concentration of TFA was <0.463 M, with the highest conversion ratio at the lowest TFA concentration tested (Table 1). Collectively, our results demonstrate that trace levels of TFA can be degraded by reductive defluorination under anoxic conditions, and by decarboxylation under oxic conditions. Assuming that our lake and saltmarsh sediments are representative of processes occurring elsewhere in the biosphere, these results suggest that the microbial flora present in most soils and sediments have the capacity to attack and degrade TFA. C.) T 75 5- _a ZIT) o_ 4 N. a N 3o E 0 _o a 0 50 100 150 200 Time (h) FIG. 2 Reductive defluorination of 2-14C-TFA in saltmarsh sediment slurries incubated without sulphate. Preparation was the same as described for Fig. 1, except the volume was increased to 80 ml slurry in 158-ml serum bottles. Samples received 5 Ci of 2-14C-TFA. Slurry subsamples (1ml) were removed by syringe and centrifuged in a microcentrifuge (15,90Og for 3 min). The supernatant was expressed through disposable 0.2 m nylon filters and kept frozen until analysis (<3 weeks). Samples were analysed by high-performance liquid chromatography with an ultraviolet absorbance detector coupled with an inline /3-emission radiation detector35. Retention times determined with unlabelled standards were (min): TFA (4.5), difluoroacetate (6.0), mono- fluoroacetate (7.5) and acetate (9.5). There was a 1.2 min delay, determined by injection of 2-14C-acetate and 2-14C-TFA, between the mass detector and the radiation detector which allowed for identification and quantification with the use of a dual-pen chart recorder. Acetate concentration was <30 M throughout the experiment (not shown). Symbols: 2-14C-TFA (O), 2-14C-difluoroacetate (A), 2-14C-monofluoroacetate (O), 2-14C-acetate (E) and 14CH4 (A). Symbols represent the mean of three individual slurries and bars indicate +1 standard deviation. Clearly, the concentration of TFA is a critical factor in this work. Our use of radiolabelled TFA enabled us to conduct experiments at concentrations that were at least four orders of magnitude lower than that employed previously by researchers using standard analytical chemical methods (F. G. Chumley, personal communication). The lowest concentration tested in our experiments was 0.185 M, based on the specific activity of the 2-14C-TFA which we added. Although this is still --1-2 orders of magnitude higher than that predicted for rainwater5.6, we feel that degradation of TFA, especially if it is a co-metabolic feature25' is likely to occur unimpeded at even lower concentra- tions. Similarly, methanogenesis is inhibited by halogenated hydrocarbons27'28 like CC14 , although anaerobic degradation of CC14 occurs when the concentration 0.5 M (refs 29, 30). Cer- tainly our results with various electron acceptors indicated a trend of higher conversion efficiencies with decreasing TFA levels (Table 1). Although our present analytical techniques pre- clude conducting experiments at TFA concentrations <0.1 M, we have no reason to question extrapolation of our results to anticipated natural concentrations. Still unanswered is the fate of TFA under conditions which favour other electron acceptors such as Fe', NO; and Mn'. However, our observation of TFA degradation under both oxic and anoxic conditions indi- cate that broadly-based microbial sinks exist for this compound in the terrestrial biosphere. Under anoxic conditions, TFA can be degraded to innocuous products like CH4 and CO2. The fact that aerobic decarboxylation of TFA yields CHF, , a gas with a long atmospheric residence time, focuses attention on the fate of this product. That methane-oxidizing bacteria can degrade methylfluoride31 as well as HFC-143 (1,1,2-trifluoroethane) (ref. 32) suggest the possibility of a soil biological sink. u Received 14 December 1993; accepted 9 May 1994. 1. Molina, M. & Rowland, F. S. Nature 249, 810-812 (1974). 2. Prather, M. J. & Watson, R. T. Nature 344, 729-734 (1992). 3. Global Ozone Research and Monitoring Project (Rep. No. 20, World Meteorological Organiz- ation, Geneva, 1989). 4. Nimitz, J. S. & Scaggs, S. R. Envir. Sci. Technol. 26, 739-743 (1992). 5. Franklin, J. Chemosphere 27, 1565-1601 (1993). 6. Rodriguez, J. M., Ko, M. K. W., Sze, N. D. & Heisey, C. W. Proc. AFEAS Workshop on Atmospheric Wet and Dry Deposition of Carbonyl and Haloacetyl Halides 25-32 (AFEAS, Washington DC, 1992). 7. Ingle, L. M. Proc. West Virginia Acad. Sci. 40, 1-11(1968). 8. Oelrichs, P. B. & McEwan, T. Nature 190, 808-809 (1961). 9. Wong, D. H., Kirkpatrick, W. E., King, D. R. & Kinnear, J. E. Soil Biol. Biochem. 24, 833838 (1992). 10. Kun, E. Citric Acid Cycle, Control and Compartmentation (ed. Lowenstein, J. M.) 279-339 (Dekker, New York, 1969). 11. Bong, C. L., Cole, A. J. L, Walker, J. R. L. & Peters, J. A. Soil Biol. Biochem. 11, 13-18 (1979). 12. Notman, P. N.Z. J. Entomol. 12, 67-71 (1989). 13. Cappenberg, T. E. & Prins, R. A. Antonie van Leeuwenhoek 40, 457-469 (1974). 14. Lovley, D. R. & Woodward, J. C. Envir. Sci. Technol. 26, 925-929 (1992). 15. Oremland, R. S., Miller, L. G. & Strohmaier, F. E. Envir. Sci. Technol. 28, 514-520 (1994). 16. Oremland, R. S., Marsh, L M. & Polcin, S. Nature 296, 143-145 (1982). 17. Oremland, R. S. & Culbertson, C. W. Nature 356, 421-423 (1992). 18. Miller, L. G., Coutlakis, M. D., Oremland, R. S. & Ward, B. B. Appl. Envir. Microbiol. 59, 2457-2464 (1993). 19. Oremland, R. S. & Capone, D. G. Adv. microb. Ecol. 10, 285-383 (1988). 20. Oremland, R. S. in Biology of Anaerobic Microorganisms (ed. Zehnder, A. J. B.) 641-706 (Wiley, New York, 1988). 21. Oremland, R. S. & Polcin, S. Appl. Envir. Microbiol. 44, 1270-1276 (1982). 22. Christensen, D. & Blackburn, T. H. Mar. Biol. 7, 113-119 (1982). 23. Mohn, W. W. & Tiedje, J. M. Microbiol. Rev. 56, 482-507 (1992). 24. Landry, K. L. & Fedorak, P. M. Arch. Microbiol. 160, 137-143 (1993). 25. Oremland, R. S. & Zehr, J. P. Appl. Envir. Microbiol. 52, 1031-1036 (1986). 26. Taylor, B. F., Hearn, W. L. & Pincus, S. Arch. Microbiol. 122, 301-306 (1979). 27. Bauchop, T. J. Bacteriol. 94, 171-175 (1967). 28. Wood, J. M., Kennedy, F. S. & Wolfe, R. S. Biochemistry 7, 1707-1713 (1968). 29. Bouwer, E. J. & McCarty, P. L. Appl. Envir. Microbiol. 45, 1295-1299 (1983). 30. Bouwer, E. J. & McCarty, P. L. Envir. Sci. Technol. 45, 1286-1294 (1983). 31. Oremland, R. S. & Culbertson, C. W. Appl. Envir. Microbiol. 58, 2983-2992 (1992). 32. DeFlaun, M. F., Ensley, B. D. & Steffan, R. J. Biotechnology 10, 1576-1578 (1992). 33. Oremland, R. S. Appl. Envir. Microbiol. 42, 122-129 (1981). 34. Culbertson, C. W., Zehnder, A. J. B. & Oremland, R. S. Appl. Envir. Microbiol. 41, 396-403 (1981). 35. Culbertson, C. W., Strohmaier, F. E. & Oremland, R. S. Origins Life Evol. Biosphere 18, 397-407 (1988). ACKNOWLEDGEMENTS. We thank F. G. Chumley, P. Crill, B. Z. Fathepure and B. F. Taylor for their comments, and E. I. du Pont de Nemours & Co. for the gift of 2-16C-TFA. This work was supported by the NASA Upper Atmosphere Research Program and the US Geological Survey. NATURE V0L 369 30 JUNE 1994 1994 Nature Publishing Group 731