Volatile fatty acids (VFAs) are important mid-products in the production of methane, and their concentrations affect the efficiency of fermentation. However, their effects on methane yield and methanogenic bacteria growth have been less extensively studied. To address these effects, acetic acid, propionic acid, butyric acid and ethanol were used as substrates and an L-9(3(4)) orthogonal table was adopted to design anaerobic digestion tests. When the highest concentrations of ethanol, acetic acid and butyric acid were 2400, 2400 and 1800 mg L-1, respectively, there was no significant inhibition of the activity of methanogenic bacteria. However, when the propionic acid concentration was increased to 900 mg L-1, significant inhibition appeared, the bacteria concentration decreased from 6 x 10(7) to 0.6-1 x 10(7) ml(-1) and their activity would not reconvert. These effects resulted in the accumulation of ethanol and VFAs, and the total methane yield consequently became very low (<321 ml). The original propionic acid concentration had a significant inhibitory effect on methanogenic bacteria growth (P < 0.01). An optimization analysis showed that ethanol, acetic acid, propionic acid and butyric acid at concentrations of 1600, 1600, 300 and 1800 mg L-1, respectively, led to the maximum accumulative methane yield of 1620 ml and the maximum methanogenic bacteria concentration of 7.3 x 10(8) ml (1). (C) 2009 Elsevier Ltd. All rights reserved.