ARTICLE Anaerobic Methanethiol Degradation and Methanogenic Community Analysis in an Alkaline (pH 10) Biological Process for Liquefied Petroleum Gas Desulfurization Robin C. van Leerdam, 1 Monica Bonilla-Salinas, 2 Frank A.M. de Bok, 3 H. Bruning, 1 Piet N.L. Lens, 1,4 Alfons J.M. Stams, 2 Albert J.H. Janssen 1 1 Department of Environmental Technology, Wageningen University, Bomenweg 2, P.O. Box 8129, 6700EV Wageningen, The Netherlands; telephone: þ31-317-482396; fax: þ31-317-482108; e-mail: robin.vanleerdam@wur.nl 2 Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands 3 Nizo Food Research, Ede, The Netherlands 4 UNESCO-IHE, Delft, The Netherlands Received 13 December 2007; revision received 19 March 2008; accepted 27 March 2008 Published online 15 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21933 ABSTRACT: Anaerobic methanethiol (MT) degradation by mesophilic (308C) alkaliphilic (pH 10) communities was studied in a lab-scale Upflow Anaerobic Sludge Bed (UASB) reactor inoculated with a mixture of sediments from the Wadden Sea (The Netherlands), Soap Lake (Central Washington), and Russian soda lakes. MT degradation started after 32 days of incubation. During the first 252 days, complete degradation was achieved till a volumetric loading rate of 7.5 mmol MT/L/day, and sulfide, methane, and carbon dioxide were the main reaction products. Temporary inhibition of MT degradation occurred after MT peak loads and in the presence of dimethyl disulfide (DMDS), which is the autooxidation product of MT. From day 252 onwards, methanol was dosed to the reactor as co-substrate at a loading rate of 3–6 mmol/L/day to stimulate growth of methylotrophic methanogens. Methanol was completely degraded and also a complete MT degradation was achieved till a volumetric loading rate of 13 mmol MT/L/day (0.77 mmol MT/gVSS/day). However, from day 354 till the end of the experimental run (day 365), acetate was formed and MT was not completely degraded anymore, indicating that methanol-degrading homoacetogenic bac- teria had partially outcompeted the methanogenic MT- degrading archea. The archeal community in the reactor sludge was analyzed by DGGE and sequencing of 16S rRNA genes. The methanogenic archea responsible for the degra- dation of MT in the reactor were related to Methanolobus oregonensis. A pure culture, named strain SODA, was obtained by serial dilutions in medium containing both trimethyl amine and dimethyl sulfide (DMS). Strain SODA degraded MT, DMS, trimethyl amine, and methanol. Flow sheet simulations revealed that for sufficient MT removal from liquefied petroleum gas, the extraction and biological degradation process should be operated above pH 9. Biotechnol. Bioeng. 2008;101: 691–701. ß 2008 Wiley Periodicals, Inc. KEYWORDS: anaerobic methanethiol degradation; salt lake sediment; methanogenesis; Upflow Anaerobic Sludge Bed (UASB) reactor; pH 10; DGGE; sequencing; LPG desulfurization Introduction Liquefied petroleum gas (LPG) is one of the top products of the crude distillation at refineries and consists mainly of light hydrocarbon compounds (C 3 and C 4 ). Light sulfur- containing compounds, such as H 2 S, methanethiol (MT) and ethanethiol (ET), are present as sulfur impurities (Bruijn, 1984; Manieh and Ghorayeb, 1981). Even though European sulfur norms for LPG do not yet exist, it is normally desulfurized to levels below 10 ppm. This study presents a new haloalkaliphilic biological process for LPG desulfurization. This novel process is an alternative for currently used physical-chemical methods like the Merox process, in which thiols are extracted from the LPG and oxidized to their corresponding disulfides that can be removed from the liquid phase (Bruijn, 1984; Verachtert et al., 1990). In the new biological desulfurization process, H 2 S and MT are removed from LPG in three steps (Sipma et al., Correspondence to: R.C. van Leerdam ß 2008 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 101, No. 4, November 1, 2008 691