Abstract The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l –1 . This strain cannot grow on any substrate under anaerobic condi- tions, but can ferment microcrystalline cellulose or sev- eral sugars to ethanol. Ethanol accumulation was eventu- ally increased, by selection and the use of a vented fer- mentation flask, to 2 g l –1 when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol, >5.0 g l –1 , were obtained by the fermentation of glucose. Little ethanol was pro- duced by the fermentation of xylose, although other fer- mentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobi- cally) a wide range of carbon sources. In addition, auxo- trophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol. Introduction The production of fuel ethanol from biomass has been considered a laudable goal because plant biomass is the only sustainable source of organic fuels, chemicals, and materials available to humanity. In most processes cur- rently under study, cellulosic biomass is first enzymati- cally hydrolyzed to sugars (often with fungal cellulases), which microorganisms (usually yeast) can then ferment to ethanol. To lower the costs associated with this pre- treatment, it is desirable to accomplish the fermentation of cellulose to ethanol in one step. In this process, termed consolidated bioprocessing (CBP) or direct mi- crobial conversion (DMC), all the steps of the conver- sion are carried out by one microorganism or a consor- tium of microorganisms in a single bioreactor (Lynd et al. 1999). Most work on CBP has concentrated on the potential of anaerobic bacteria to convert cellulosic bio- mass to ethanol. However, some filamentous fungi hold promise in this area, and there are some advantages to fungi as the mediators of biomass conversion. They do not require strictly anaerobic conditions and so can be directly inoculated onto cellulosic biomass, and their fil- amentous growth habit facilitates separation of cell mass from the broth. Also, many fungal strains produce copi- ous numbers of conidiospores (conidia), which could be useful for inoculation at a high level, making the inocu- lation of non-sterile biomass more practical. Several fila- mentous fungi have been reported to directly ferment cellulose to ethanol, though on rich, undefined media. These include members of the genera Aspergillus, Rhizo- pus (Skory et al. 1997), Monilia (Gong et al. 1981), Neu- rospora (Deshpande et al. 1986), and Fusarium (Singh and Kumar 1991). In this study, environmental samples were screened for organisms capable of producing ethanol from cellu- lose on minimal medium. We have isolated a filamentous fungus capable of this conversion, and have made some progress toward increased ethanol yields and develop- ment of a genetic system based on parasexuality. Disclaimer: Use of commercial products does not imply an endorse- ment or warranty of such products by the USDA. D.M. Stevenson ( ✉ ) · P.J. Weimer U. S. Dairy Forage Research Center, 1925 Linden Drive West, Madison, WI 53706, USA and Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA e-mail: DMStevenson@wisc.edu Tel.: +1-608-2645320, Fax: +1-608-2645147 Appl Microbiol Biotechnol (2002) 59:721–726 DOI 10.1007/s00253-002-1027-3 SHORT CONTRIBUTION D. M. Stevenson · P. J. Weimer Isolation and characterization of a Trichoderma strain capable of fermenting cellulose to ethanol Received: 3 December 2001 / Revised: 8 April 2002 / Accepted: 19 April 2002 / Published online: 19 July 2002 © Springer-Verlag 2002