Anoxic Bioremediation of Hydrocarbons

scientific correspondence curiously spongy, represents the earliest strain JS-150 and amended with chlorite record of perichondral, and therefore endo- a under anaerobic conditons, [14C]naphtha- 70 14 skeletal, bone in a primitive chondrich- lene is rapidly oxidized to CO2 (data not 11 14 thyan. With few exceptions , the absence of 60 shown). No CO2 is produced if either of endoskeletal bone is seen as a defining fea- 50 the organisms or chlorite is omitted, unless 8 ture of extant chondrichthyans , relative to C] benzene) O2 is added to the headspace. The degrada- 14 40 its primitive presence in other jawed and tion of naphthalene is therefore directly fossil jawless vertebrates. This unexpected 30 dependent on the combined presence of 20 Coal tar pit sediment juxtaposition of bone and GCC may repre- Pristine soil both strain CKB and chlorite. 8 sent a further, persistent, primitive aspect of 10 Coal tar pit negative control Because strain CKB cannot degrade aro-

(% of initial [ Pristine soil negative control early chondrichthyan skeletal tissues. 2 0 matic hydrocarbons in pure culture, we con-

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Alternatively, the near-continuity of this 14 sidered the possibility that the stimulation of bone layer with the fin spine could indicate Time (day) hydrocarbon degradation could be the result an unusual distribution of dermal skeleton of chlorite dismutation into chloride and O2 b derived from the trunk or cranial neural Contaminating by strain CKB. The resultant O2 is used by CO crest12. Finally, the disputed relationship of hydrocarbons 2 indigenous, aerobic hydrocarbon-oxidizing 1,5 Hydrocarbon- — + the brush to the fins is resolved by regard- oxidizer 4e +4H bacteria that are otherwise inhibited by the ing it as a specialized fin–baseplate exten- anoxic condition of the soil (Fig. 1b). In sup- — — 2H2O sion. Its endoskeletal location, histology ClO2 Cl + O2 port of this, when chlorite is amended to an and absence of fin radials support this idea. anaerobic, washed whole-cell suspension of M. I. Coates*, S. E. K. Sequeira*, Strain CKB strain CKB, O2 is rapidly and proportionally I. J. Sansom†, M. M. Smith‡ produced. There is no O2 production if the *Biology Department, Figure 1 Stimulation of aromatic hydrocarbon oxida- cells are omitted or killed by heat. University College London, tion by chlorite dismutation. a, Oxidation of [14 C]- Our results show that the dismutation 14 London WC1E 6BT, UK benzene to CO2 in anoxic contaminated and of chlorite by perchlorate-reducing bacteria e-mail: [email protected] pristine soil and sediment samples amended with in anaerobic environments can produce

†School of Earth Sciences, chlorite and inoculated with strain CKB. Arrowheads, extracellular O2. This O2 can be used by University of Birmingham, points of addition of 1 mM chlorite. b, Mechanism hydrocarbon-oxidizing bacteria to degrade Birmingham B15 2TT, UK by which chlorite dismutation might stimulate hydro- hydrocarbons, such as benzene, which is a ‡Department of Craniofacial Development, carbon oxidation in anoxic sediments. particularly important environmental cont- School of Dentistry, aminant owing to its toxicity and relative UMDS Guy’s Hospital Campus, of chlorite into molecular oxygen and chlor- solubility. Little is known about perchlorate- London SE1 9RT, UK ide is an intermediate step in the microbial reducing bacteria, although they are ubiqui- 4 1. Zangerl, R. J. Vert. Paleontol. 4, 372–378 (1984). reduction of perchlorate or chlorate . tous in a wide range of environments, 2. Maisey, J. G. Zool. J. Linn. Soc. 66, 161–183 (1979). As part of a study on microbial perchlor- including pristine soils and petroleum- 5 3. Smith, M. M. & Hall, B. K. Biol. Rev. 65, 277–373 (1990). ate reduction, we isolated a new micro- contaminated sediments . 4. Patterson, C. Phil. Trans. R. Soc. Lond. B 249, 101–205 (1965). organism, strain CKB, which grows High concentrations of chlorite may be 5. Lund, R. Ann. Carnegie Mus. 45, 161–178 (1974). 6. Williams, M. E. Paleontographica 190, 83–158 (1985). anaerobically by reducing perchlorate or toxic to many microbial species, but our 7. Wood, S. P. Nature 297, 574–577 (1982). chlorate, from waste sludge from a paper results indicate that significant degradation 8. Janvier, P. Early Vertebrates (Oxford Univ. Press, 1996). mill in Pennsylvania. When strain CKB is of hydrocarbons can be stimulated at chlor- 9. Ørvig, T. Arkiv Zool. 2, 321–454 (1951). inoculated with chlorite into anoxic, ite concentrations (1 ȖM is 90 Ȗg per litre) 10.Sansom, I. J. et al. Nature 379, 628–630 (1996). 14 11.Peignoux-Deville, J., Lallier, F. & Vidal, B. Cell Tissue Res. 222, petroleum-contaminated soil samples, [ C]- well below the limits imposed by the World 14 Ȗ 605–614 (1982). benzene is rapidly oxidized to CO2, with Health Organization (200 g per litre) and 14 12.Smith, M. M. et al. Proc. R. Soc. Lond. B 256, 137–145 (1994). about 40% of the original C being recov- the US Environmental Protection Agency ered in this form after two days of incuba- (1 mg per litre)6,7. As a bioremediative strat- tion (Fig. 1a). If the sediments are further egy, the application of chlorite dismutation amended with 1 mM chlorite on day 3, to stimulate hydrocarbon oxidation in Anoxic bioremediation about 60% of the 14C can be recovered as contaminated environments offers a new 14 14 CO2 by day 6 (Fig. 1a). No CO2 is pro- alternative to other injection processes. of hydrocarbons duced in samples that are not amended John D. Coates, Royce A. Bruce, with chlorite or strain CKB. John D. Haddock The contamination of soils and sediments Similar results are obtained with anoxic Department of Microbiology, by petroleum is a matter of international soil samples that have had no previous Southern Illinois University, concern because of the toxicity and refrac- exposure to hydrocarbons (Fig. 1a). How- Carbondale, Illinois 62901, USA tory character of the aromatic components ever, there is a slight lag phase of 24 hours, e-mail: [email protected] 1 in the absence of oxygen . Gaseous oxygen which is consistent with adaptation to ben- 1. Anderson, R. T. & Lovley, D. R. Adv. Microbiol. Ecol. 15, can be injected into the anaerobic zone of a zene by the microbial population. The stim- 289–350 (1997). contaminated environment2 to stimulate ulatory effects are not significantly altered 2. Crocetti, C. A., Head, C. L. & Ricciardelli, A. J. Aeration-Enhanced Bioremediation of Oil-Contaminated Soils: A Laboratory Treatability biodegradation, but this is costly and inef- when lower chlorite concentrations are Study (National Groundwater Association, Houston, 1992). ficient. Other more soluble electron accep- used: 1 ȖM chlorite resulted in more than 3. Coates, J. D., Anderson, R. T. & Lovley, D. R. Appl. Environ. tors, such as nitrate or sulphate, can be half the level of benzene degradation Microbiol. 62, 1099–1101 (1996). used instead, but oxidation is slow and observed with 1 mM chlorite. 4. Rikken, G., Kroon, A. & van Ginkel, C. Appl. Microbiol. 3 Biotechnol. 45, 420–426 (1996). hydrocarbon degradation is incomplete . This stimulatory effect also occurs in 5. Michaelidou, U., Bruce, R., Achenbach, L. & Coates, J. D. in Here we describe how chlorite dismutation defined mixed cultures in the absence of Abstr. 98th Gen. Meeting Am. Soc. Microbiol. 313 (American by perchlorate-reducing bacteria can be soil. When an anaerobic, washed-cell sus- Society for Microbiology, Atlanta, 1998). 6. Foundation for Water Research Research Report FR0390 (1993). used as an alternative source of oxygen for pension of strain CKB is combined with the 7. US Environmental Protection Agency Fed. Reg. 59, 6331–6444 degrading contaminants. This dismutation aerobic hydrocarbon-oxidizing Pseudomonas (1994).