Butanol (butyl alcohol or n-butanol) is a four carbon straight chained alcohol which can be usedrnas a liquid transportation biofuel. The objective of this study was production of bio butanol fromrnbrewery spent grain (BSG) which in effects to minimize energy cost and substituting nonrenewablernenergy by using renewable resources. The chemical and proximate compositions analysis wasrninvestigated. The moisture, ash, extractives, hemicellulose, lignin and cellulose contents were,rn64.65, 4.2, 33.28, 17.79 and 17.87 wt. % respectively. The conversion of BSG to bio butanol canrnbe achieved mainly by four process steps,pre-treatment, hydrolysis (dilute acid hydrolysis),rnfermentation and separation. Dilute acid hydrolysis was employed, because it is easy andrnproductive process. Fermentation of the hydrolyzate was performed using 10 % v/vrnClostridium/Eubacterium at 37rnC temperature, pH 6.5 and 96 hr fermentation time for all samples.rnThe experiment was designed by Box-Behnken Design (BBD) using Design expert 7.0.0 softwarernwith three factors to investigate the effect of (temperature, reaction time and acid concentration).rnHydrolysis temperature was varied from 115Â°C to 135â„ƒ, reaction time was varied from 30 to 40rnminutes and the acid concentration was varied from 1.5% to 2%. Significance of the processrnvariables were analyzed using analysis of variance (ANOVA) and the quadratic model was fittedrnto the experimental results. Thus, the influence of all experimental variables, factors andrninteraction effects on the response was investigated. Among the investigated componentsrnhydrolysis temperature, reaction time, acid concentration, interaction between temperature andrnreaction time, interaction between temperature and acid concentration contributed a significantrneffect on the yield of bio butanol. As the result of RSM optimization, the best yield of total reducingrnsugar (TRS) and bio butanol were found at 127.42rn0rnC hydrolysis temperature, 34.32min reactionrntime and 1.65% w/w acid concentration. Under these conditions, 59.0 g/L total reducing sugarrn(TRS), 10.93 g/L biobutanol yield and 0.11 g/L.h of productivity were obtained. The optimizedrnprocess variables were farther enhanced by co culturing Clostridium/Eubacterium and Bacillusrnsubtilis. Different inoculum concentration ratios (10:1%, 10:3%, and 10:5% v/v) ofrnClostridium/Eubacterium to Bacillus subtilis) were used to ferment BSG hydrolysate. The resultsrnexhibited that the inoculum size of 10% Clostridium/Eubacterium with 1% Bacillus subtilis rnmaximised the production of biobutanol to 13.76g/L and 0.14 g/L.h productivity.