Appl Microbiol Biotechnol (2004) 64: 421–428 DOI 10.1007/s00253-003-1430-4 ENVIRONMENTAL BIOTECHNOLOGY J. Sipma . R. J. W. Meulepas . S. N. Parshina . A. J. M. Stams . G. Lettinga . P. N. L. Lens Effect of carbon monoxide, hydrogen and sulfate on thermophilic (55°C) hydrogenogenic carbon monoxide conversion in two anaerobic bioreactor sludges Received: 14 April 2003 / Revised: 21 July 2003 / Accepted: 26 July 2003 / Published online: 11 October 2003 # Springer-Verlag 2003 Abstract The conversion routes of carbon monoxide (CO) at 55°C by full-scale grown anaerobic sludges treating paper mill and distillery wastewater were elucidated. Inhibition experiments with 2-bromoethane- sulfonate (BES) and vancomycin showed that CO con- version was performed by a hydrogenogenic population and that its products, i.e. hydrogen and CO 2 , were subsequently used by methanogens, homo-acetogens or sulfate reducers depending on the sludge source and inhibitors supplied. Direct methanogenic CO conversion occurred only at low CO concentrations [partial pressure of CO (P CO ) <0.5 bar (1 bar=10 5 Pa)] with the paper mill sludge. The presence of hydrogen decreased the CO conversion rates, but did not prevent the depletion of CO to undetectable levels (<400 ppm). Both sludges showed interesting potential for hydrogen production from CO, especially since after 30 min exposure to 95°C, the production of CH 4 at 55°C was negligible. The paper mill sludge was capable of sulfate reduction with hydrogen, tolerating and using high CO concentrations (P CO >1.6- bar), indicating that CO-rich synthesis gas can be used efficiently as an electron donor for biological sulfate reduction. Introduction Carbon monoxide (CO) can support a complex microbial food chain of a variety of trophic groups. CO can be metabolized by methanogens or by sulfate reducers, possibly via H 2 and CO 2 as intermediates (Mörsdorf et al. 1992). Hydrogenogens (Svetlichnyi et al. 2001) are capable of converting CO into H 2 and CO 2. Furthermore, acetogens are reported to convert CO into acetate (Mörsdorf et al. 1992) or ethanol and butanol (Bredwell et al. 1999). The latter compounds can be utilized by other anaerobes. Table 1 presents reactions that are possibly involved in the anaerobic conversion of CO and summarizes their stoichiometry and Gibbs free energy under standard conditions (25°C) and 55°C. In view of the current interest in biohydrogen, the biological conversion of CO with production of hydrogen is a very promising reaction. Biohydrogen production can support the transition of the current unsustainable fossil fuel based energy economy into a hydrogen economy. Furthermore, H 2 has a great potential in (bio)chemical processes as an electron donor in various chemical and biotechnological reductive processes, e.g. in biological sulfate reduction processes (van Houten et al. 1994). A cheap source of hydrogen -rich gas is synthesis gas, which is produced by, for example, steam reforming of natural gas, or thermal gasification of coal, oil, biomass or other organic matter. The composition of the H 2 -rich synthesis gas produced varies greatly and depends on the source material gasified and the gasification method employed (Perry et al. 1997). The major restriction of synthesis gas utilization is the presence of CO, which can range from 5% to over 50% (Perry et al. 1997). Sulfate reduction with synthesis gas is hampered by the presence of CO concentrations of as low as 5% (van Houten et al. 1996a). In proton exchange membrane fuel cells, less than 20 ppm CO can be tolerated (Otsuka et al. 2002). Microorganisms capable of converting CO into hydrogen could represent an interesting biological alternative to the currently employed chemical catalytic water-gas shift reaction (reaction 1, Table 1), especially since biological J. Sipma (*) . R. J. W. Meulepas . G. Lettinga . P. N. L. Lens Sub-department of Environmental Technology, Wageningen University, Bomenweg 2, P.O. Box 8129, 6700 EV Wageningen, The Netherlands e-mail: jan.sipma@wur.nl Tel.: +31-317-485098 Fax: +31-317-482108 S. N. Parshina Laboratory of Microbiology of Anthropogenic Environments, Institute of Microbiology Russian Academy of Sciences, Prosp. 60 let Oktyabrya, 7-2, 117811 Moscow, Russia A. J. M. Stams Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands