Whiskey distillery waste streams consisting of pot ale (liquid residue) and spent grain (solid residue) are high strength organic wastes and suitable feedstock for anaerobic digestion (AD) from both economic and environmental stand points. Anaerobic digestion of pot ale and pot ale/spent grain mixtures (with mixing ratios of 1:1, 1:3, and 1:5 by wet weight) was performed after implementation of a novel hybrid pre-treatment (combined chemical and mechanical) in order to modify lignocellulosic structure and ultimately enhance digestion yield. Lignin, hemicellulose, and cellulose fractions were determined before and after chemical pre-treatment. Effects of different inoculum rates (10-30-50% on wet basis) and beating times (0-7.5-15 min) on anaerobic digestion of pot ale alone and of pot ale/spent grain mixtures were investigated in lab scale batch mode with a major focus of optimising biogas yield by using response surface methodology (RSM) in Design Expert Software. The highest biogas yields of 629 ± 8.5 mL/g vs. (51.3% CH 4 ) and 360 ± 10 mL/g vs. (55.0 ± 0.4) with anaerobic digestion of pot ale alone and spent grain mix after 1M NaOH and 7.5 min beating pre-treatments with 50% inoculum ratio respectively. The optimum digestion conditions to maximise the biogas quality and quantity were predicted as 10 and 13 min beating times and 32 and 38 • C digestion temperatures for anaerobic digestion of pot ale alone and spent grain mix respectively.Processes 2020, 8, 492 2 of 27 of alternative energy sources has arisen [5]. Among the renewable energy sources, biogas, which is methane rich gas produced by anaerobic digestion (AD) of organic waste, is considered to be among the most environmentally friendly fuel sources [6] owing to its non-toxic characteristics and potential for simplicity of use as an alternative over traditional fossil fuels, as well as its potential reduction in wastes sent to landfill [7].Typical waste disposal levels of whiskey distilleries and breweries and the potential environmental concerns associated with those waste streams was outlined by [8]. Due to the high organic content, large disposal volume, and the acidic nature, pot ale is the major concern of the waste management of whiskey distilleries. As such, AD with advantages over other waste management methods like low energy requirement, odour limitation, less by-product generation, and higher ability to cope with recalcitrant nature of whiskey distillery/brewery waste streams is a suitable alternative waste treatment method [9][10][11][12]. There are four main stages in the AD process-namely hydrolysis, acidogenesis, acetogenesis, and methanogenesis-and the reactions in each stage are carried out by different bacteria. The thermodynamics of each AD stage and potential bacterial competitions have been reported in detail by [13,14]. In order to have a balanced AD, identical reaction rates are desired, however, the hydrolysis step is considered to be the rate limiting step for the digestion of lignocellulosic matter due to the impact of high lig...