Perturbation of Syntrophic lsobutyrate and Butyrate Degradation with Formate and Hydrogen Wei-Min Wu,' Mahendra K. Jain,"** Robert F. Hickey,' and J. Gregory Zeikuslr3 zyxwvutsr 'MBI International, 3900 Collins Road, P. 0. Box 27609, Lansing, Michigan 48909; and 'Department of Animal Science and 3Department of Biochemistry and Microbiology, Michigan State University, East Lansing, Michigan 48824 Received November 14, 1995/Accepted April 9, 1996 zyxwv The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and isobutyrate was investigated using a defined methanogenic culture, con- sisting of syntrophic isobutyrate-butyrate degrader strain IB, Methanobacterium formicicum strain TIN, and Methanosarcina mazeii strain T18. Formate and hydro- gen were used to perturb syntrophic butyrate and isobu- tyrate degradation by the culture. The reversible isomer- ization between isobutyrate and butyrate was inhibited by the addition of either formate or hydrogen, indicating that the isornerization was coupled with syntrophic bu- tyrate degradation for the culture studied. Energetic analysis indicates that the direction of isomerization be- tween isobutyrate and butyrate is controlled by the ratio between the two acids, and the most thermodynamically favorable condition for the degradation of butyrate or isobutyrate in conjunction with the isomerization is at almost equal concentrations of isobutyrate and butyrate. The degradation of isobutyrate and butyrate was com- pletely inhibited in the presence of a high hydrogen par- tial pressure b2000 Pa) or a measurable level of formate (10 zyxwvutsrqp pM or higher). Significant formate (more than 1 rnM) was detected during the perturbation with hydrogen (17 to 40 kPa). Resumption of butyrate and isobutyrate deg- radation was related to the removal of formate. Energetic analysis supported that formate was another electron carrier, besides hydrogen, during syntrophic isobutyrate- -butyrate degradation by this culture. 0 1996 John Wiley & Sons, Inc. Key words: anaerobic digestion isobutyrate butyrate isomerization hydrogen formate INTRODUCTION zyxwvutsrq Butyrate and isobutyrate are important intermediates which are observed during anaerobic digestion (McInerney, 1988). A reversible isomerization between butyrate and isobutyrate occurs during butyrate or isobutyrate degradation (Tholozan et al., 1988). To date, most research on isobutyrate degra- dation was performed using undefined, mixed cultures such as lake sediments (Lovley and Klug, 1982), digester sludges (Aguilar et al., 1990; Zinder et al., 1984), isobutyrate en- * To whom all correspondence should be addressed. Telephone: (517)336-4626, fax: 517-337-2122; e-mail: jain@mbi.org richments (Stieb and Schink, 1989; Tholozan et al., 1988), and methanogenic granules (Wu et al., 1993b). Matthies and Schink (1992) have reported anaerobic degradation of iso- butyrate by a defined culture consisting of strain WoG13, zy Syntrophomonas wolfeii, and Methanosprillum hungateii. In this culture, however, strain WoG13 did not degrade isobu- tyrate but only isomerized isobutyrate to butyrate that was then degraded by S. wolfeii, a butyrate degrader, together with Msp. hungateii to acetate and methane (Matthies and Schink, 1992). A defined syntrophic isobutyrate-butyrate degrading cul- ture has been isolated, which consisted of a gram-positive, non-spore-forming, isobutyrate and butyrate degrading rod strain IB, a H,/formate utilizing Methanobacterium formici- cum strain TlN, and an acetate/€€,-utilizing Methunosar- cina mazeii strain T18 (Wu et al., 1992, 1994). This culture degraded isobutyrate, butyrate, and other normal and branched fatty acids with four to nine carbon atoms to meth- ane and CO,. During degradation of butyrate or isobutyrate by this culture, isobutyrate, or butyrate was always isomer- ized to the other isomeric form to reach near equal concen- trations. The strain IB perhaps converts isobutyrate to bu- tyrate and then degrades the latter to acetate. Therefore, this culture can serve as a tool to investigate the mechanism of isomerization and syntrophic isobutyrate-butyrate degrada- tion. During syntrophic degradation of ethanol and saturated fatty acids with three or more carbon atoms, H, has been detected as an intermediate/electron carrier (Boone and Bry- ant, 1980; Bryant, 1979; McInerney et al., 1980, 1981). High concentrations of H, completely inhibited syntrophic butyrate degradation (Lovley and Mug, 1982; McInerney et al., 1980). Formate was also proposed to be an intermediate/ electron carrier of syntrophic ethanol degradation (Ozturk et al., 1989; Thiele and Zeikus, 1988) and butyrate degrada- tion (Bae and McCarty, 1993; Boone et al., 1989). The importance of formate as an electron carrier was questioned in some methanogenic granular sludges containing abun- dant Methanobrevibacter arboriphilus-like methanogens, which did not utilize formate (Grotenhuis et al., 1991). Both hydrogen and formate were detected as intermediate prod- Biotechnology and Bioengineering, Vol. 52, Pp. 404-41 1 (1996) 0 1996 John Wiley &Sons, Inc. CCC 0006-3592/96/030404-08