Production of medium-chain volatile fatty acids by mixed ruminal microorganisms is enhanced by ethanol in co-culture with Clostridium kluyveri Paul J. Weimer a,b,⇑ , Michael Nerdahl b , Dane J. Brandl b a US Dairy Forage Research Center, Agricultural Research Service, United States Department of Agriculture, Madison, WI 53706, USA b Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA highlights  Clostridium kluyveri 3231B grew well with mixed ruminal bacteria in vitro.  Co-cultures fermented mixtures of cellulosic biomass and ethanol to C 4 –C 6 VFAs.  Co-cultures produced up to 6.1 g caproate and 1.6 g valerate L 1 in 48 h at 39 °C.  This VFA production system represents a useful extension of the carboxylate platform. article info Article history: Received 23 September 2014 Received in revised form 10 October 2014 Accepted 12 October 2014 Available online 18 October 2014 Keywords: Caproic acid Carboxylate platform Clostridium kluyveri Ethanol Rumen abstract Mixed bacterial communities from the rumen ferment cellulosic biomass primarily to C 2 –C 4 volatile fatty acids, and perform only limited chain extension to produce C 5 (valeric) and C 6 (caproic) acids. The aim of this study was to increase production of caproate and valerate in short-term in vitro incubations. Co- culture of mixed ruminal microbes with a rumen-derived strain of the bacterium Clostridium kluyveri converted cellulosic biomass (alfalfa stems or switchgrass herbage) plus ethanol to VFA mixtures that include valeric and caproic acids as the major fermentation products over a 48–72 h run time. Concen- trations of caproate reached 6.1 g L 1 , similar to or greater than those reported in most conventional carboxylate fermentations that employ substantially longer run times. Published by Elsevier Ltd. 1. Introduction The carboxylate platform for biofuels production is a means of converting organic wastes and biomass to organic acids, particu- larly volatile fatty acids (VFA) using undefined mixed cultures of anaerobic microorganisms typically derived from sewage sludge digesters or aquatic sediments (Agler et al., 2011; Chang et al., 2010). In the carboxylate platform, a conventional anaerobic diges- tion process is altered via pH control or addition of methanogenic inhibitors, upon which accumulating C 2 –C 4 VFA are extended to medium chain VFA (C 5 –C 8 ) via reverse b-oxidation (RBO) reactions. The medium chain VFA can be recovered by extraction or other methods (Singhania et al., 2013) and converted by catalytic chemistry or electrochemistry to various liquid fuels or industrial chemicals. Examples of potential products include ketones and secondary alcohols produced in the MixAlco process (Holtzapple and Granda, 2009); esters by a process analogous to the valeric bio- fuels platform of Lange et al. (2010); or hydrocarbons and alcohols produced by Kolbe or Hoefer–Moest electrolysis (Levy et al., 1983). The carboxylate platform has several attractive features, including feedstock flexibility; minimal feedstock pretreatment; utilization of most organic components of the biomass (carbohydrate, protein, nucleic acid, organic acids and some lipids); non-aseptic operation; and mixed culture stability. The main limitation of the biological (fermentation) component of the carboxylate platform based on anaerobic digester communities is the slow rate of VFA chain extension (incubation times of weeks). As an alternative to the above configuration, biomass may be fermented in bioreactors using mixed cultures of microbes from the rumen. These ‘‘extraruminal’’ fermentations have some addi- tional advantages over the originally described carboxylate plat- form (Weimer et al., 2009). Fermentation run times are http://dx.doi.org/10.1016/j.biortech.2014.10.054 0960-8524/Published by Elsevier Ltd. ⇑ Corresponding author at: USDA-ARS-USDFRC, 1925 Linden Drive West, Madison, WI 53717, USA. Tel.: +1 608 890 0075. E-mail address: Paul.Weimer@ars.usda.gov (P.J. Weimer). Bioresource Technology 175 (2015) 97–101 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech