The effect of enzyme addition on anaerobic digestion of Jose Tall Wheat Grass Rowena T. Romano a , Ruihong Zhang a, * , Sarah Teter b , Jeffery A. McGarvey c a Department of Biological and Agricultural Engineering, University of California, 2030 Bainer Hall, One Shields Avenue, Davis, CA 95616-5294, USA b Novozymes Inc., Davis, CA 95616, USA c USDA/ARS/FCR, Albany, CA 94710, USA article info Article history: Received 26 November 2007 Received in revised form 22 December 2008 Accepted 25 December 2008 Available online 24 May 2009 Keywords: Cellulase Wheat grass Anaerobic digestion 16S rRNA abstract The effects of the addition of enzyme products containing cellulase, hemicellulase, and b-glucosidase to anaerobic digestion systems were studied using Jose Tall Wheat Grass (wheat grass) as a model substrate. Anaerobic digestion tests were performed using batch reactors operated at 50 °C. The application of enzyme products in three digestion configurations were simulated and investigated: (1) enzyme addition to a single-stage digester, (2) pre-treatment of wheat grass with enzymes followed by a single-stage anaerobic digestion, and (3) enzyme addition to the first stage (hydrolysis and acidification) of a two- stage digestion system. The enzyme products showed positive effects on the solubilization of wheat grass when used alone to treat the wheat grass. However, no significant differences in biogas and methane yields, and volatile solids reduction resulted when the enzyme products were tested in the anaerobic digestion systems. This reveals that the microorganisms present in the inoculum were effective in carry- ing out the digestion of wheat grass. The types of microorganisms present in the inoculum were identi- fied using 16S rRNA sequence analysis. A comparison of the sequences between the different inocula revealed that the prevalent operational taxonomic units were similar, but that the acidified inoculum contained a higher percentage of the species Thermotogae. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Anaerobic digestion is an attractive technology for the treat- ment of organic waste (e.g., manure, food-processing wastes and green wastes). The microorganisms within an anaerobic digester work synergistically to convert organic matter into biogas (meth- ane (CH 4 ) and carbon dioxide (CO 2 ) that has an energy content ranging from 18,630 to 26,081 kJ/m 3 depending on the CH 4 con- tent. Biological degradation of lignocellulosic material is normally facilitated by enzymes, such as cellulases and hemicellulases, which are produced by the microorganisms. The rate-limiting step for anaerobic digestion of lignocellulosic material is the hydrolysis of cellulose and hemicellulose. Increasing the hydrolysis rate is critical in order to improve the biomass-conversion efficiency of anaerobic digestion. Previous studies have reported that adding enzymes into anaer- obic digesters treating food-processing wastes resulted in im- proved digestion. Akao et al. (1992) reported enhanced anaerobic digestion of citrus peels with an enzyme solution from Aspergillus sp. A-1. The enzyme solution was reported to have cellulase and pectinase activities that allowed the anaerobic digester to operate at a higher organic loading rate. In another study, Sonakya et al. (2001) pretreated wheat grains with Trizyme (cellulase, a-amy- lase, and protease) prior to anaerobic digestion and observed an in- crease in methane production by 7–14%. The addition of cellulase (Celluclast 200L) and b-glucosidase (Novozyme 188) to anaerobic digesters treating sewage sludge was reported by Higgins and Swartzbaugh (1986). The system consisted of an enzyme pretreat- ment stage followed by anaerobic digestion and resulted in in- creased biogas and methane yields of 12% and 15%, respectively. In another study, Nagle et al. (1992) treated previously-digested sewage sludge with protease K (type XI, from Tritirachium album), thermolysin (type X from Bacillus thermoproteolyticus), trypsin (type XI, from bovine pancreas), and lysozyme (from chicken egg white), which resulted in a 22% increase in soluble chemical oxy- gen demand (SCOD) after a 4.1 h treatment. Rintala and Ahring (1994) investigated addition of Pulpzyme HA (xylanse and cellu- lase), Alcalase 2.5 LB (protease), and Resinase A 2X (lipase) to the anaerobic digestion of source-separated household solid waste. In batch treatments, enzymes were added individually or as a mix- ture, and the specific methane activity (SMA) was measured (de- fined as the slope of the cumulative methane production from the initial 20–30 h of digestion per volatile solids (VS) content of the inoculum). Only the lowest concentration of protease applied (0.5 ml/kg VS) resulted in significantly higher (11%) SMA. However, the authors reported no significant differences in biogas yield or 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.12.065 * Corresponding author. Tel.: +1 530 752 9530; fax: +1 530 752 2640. E-mail address: rhzhang@ucdavis.edu (R. Zhang). Bioresource Technology 100 (2009) 4564–4571 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech