Biofuels obtained from first generation (1G) sugars-starch streams have been proven unsustainable as their constant consumption is not only significantly costly for commercial scale production systems but it could potentially lead to problems associated with extortionate food items for human usage. The valorization of second generation (2G) lignocellulosic biomass through bio-fermentation employing extremophiles is the strategic tool leading to sustainable process development for biofuels’ generation. In this regard, sugarcane bagasse (SCB) is a potential valuable lignocellulosic biomass, adequate enough in carbon for fermentative processes and embodying itself as waste, whose disposal is considered as burden on natural environment especially in agricultural countries like Pakistan. 2G fermentation technologies at large scale for ethanol and hydrogen productions are limited due to low reactor productivity, product titer and yield. In the present investigation, a moderate alkali-thermophilic ethanologenic bacterium was isolated from soil sampled from the vicinity of hot water effluent near Balkasar oil refinery, Chakwal, Pakistan and identified through 16S rRNA gene sequencing as Bacillus licheniformis. Consequently, it was allotted the accession No. KU886221. The isolate was found to ferment glucose and xylose thus making it a potential candidate to employ SCB for ethanologenesis. Further valuation on the ethanologenic potential of the isolate was done by using SCB pretreated with H2SO4, H3PO4, HCl and NaOH following with and without enzymatic hydrolysis in the fermentation medium. The outcome revealed that significantly elevated level of ethanol was achieved in the fermentation medium by using SCB hydrolysate (SCBH) obtained after enzymatic hydrolysis of alkaline pretreated SCB. A standard 2-factor central composite response surface design was used to estimate the optimized concentration of cellulose and hemicellulose degrading enzymes (1.024 g Cellic Ctec + 0.468 g Htec Novozymes/100 g of pretreated SCB with 1N NaOH) for breaking down alkaline pretreated SCB to maximum fermentable sugars. To economize the expenditure associated with biofuels production, the low-cost corn steep liquor (CSL), a chief by-product of corn starch processing was used as nitrogen source. It was found that 3.24 % more ethanol was produced when CSL was used in the fermentation medium substituting peptone + yeast extract. Six factors (concentration of pretreated SCB hydrolysates, CSL, sodium chloride, incubation temperature, pH and fermentation period) affecting ethanol fermentation were optimized using Taguchi OA L27 (3^13) of Design-Expert 8 software. A batch-culture was carried out under optimized conditions for ethanol fermentation in bench-scale stirred-tank bioreactor. The ethanol titer was 11.301 g/L corresponding 0.909 mol of ethanol/mol of sugars consumed with 98.5 % reduction of substrate. In terms of biomass, 0.114 g ethanol/g of alkaline pretreated SCB was obtained. Next, the fed batch fermentation was performed to understand to consequence of substrate addition on batch fermentation. Significantly improved level of ethanol was found at the end of fermentation (120 hours) i.e.16.896 g/L corresponding 0.973 mol ethanol/mol sugars consumed. In terms of biomass, 0.123 g ethanol/g of alkaline pretreated SCB was obtained at the end of fed batch fermentation experiment. Further modification in fed batch fermentation setup was done by incorporating immobilized cells fibrous-bed bioreactor (FBB) to surpass the production of ethanol under alkali-thermophilic fermentation conditions. Subsequently, improved ethanol production associating 85.031 % substrate utilization was observed with 1.076 mol ethanol/mol of sugars consumed. Ethanol titer significantly increased from 16.896 to 19.39 g/L with the attachment of FBB. In terms of biomass, 0.131 g ethanol/g of alkaline pretreated SCB was achieved at the end of fermentation. Furthermore, to alleviate the effect of ethanol induced inhibition on ethanol fermentation, the in situ gas stripping was performed during the fermentation through the culture medium. Highest ethanol titer of 21.637 g/L equivalent to 1.1406 mol ethanol/mol of sugars consumed with 94.295 % substrate consumption was obtained. In terms of biomass 0.135 g ethanol/g of alkaline pretreated SCB was obtained at the end of fed batch fermentation involving FBB and gas stripping. Finally, fed batch fermentation involving FBB was studied under non-aseptic conditions. The results demonstrated that comparing aseptic conditions, 30.5 % reduced ethanol was obtained under non-aseptic conditions showing the likelihood of some contaminant(s) in the fermentation culture. In the second part of investigation, Clostridium thermocellum DSMZ 1313, a renowned thermophilic cellulolytic bacterium was used for co-production of bioethanol and biohydrogen employing sugarcane bagasse directly as fuels’ feed. Six factors (cellulose, CSL, iron sulphate, magnesium chloride, incubation pH and period) affecting co-fermentation of bioethanol and biohydrogen were optimized using Taguchi OA experimental design. In the following experiment, cellulose was substituted with SCB pretreated with different chemicals in separate batch experiments. It was determined that SCB pretreated with 2 % H2SO4 produced significantly greater amounts of bioethanol and biohydrogen under optimized fermentation conditions in the fermentation medium. Batch fermentation in bench-scale stirred-tank bioreactor was performed under aseptic conditions by rotating the fermentation conditions elucidated by Taguchi OA favoring maximum production of both fuels. The fermentation yielded 1.027 mol of ethanol/mol of equivalent sugar with ethanol titer of 8.662 g/L and 0.775 mol of hydrogen/mol of equivalent glucose consumed with hydrogen titer of 2.97 L/L of fermentation medium at end of the experiment with 73.081 % substrate reduction. In terms of biomass, batch yields were 0.101 g ethanol/g of biomass and 34.715 mL of hydrogen/g of biomass utilized. Afterward, FBB was incorporated in the bioreactor to enhance substrate utilization. The percent substrate consumption increased to 93.837 whereas ethanol yield decreased to 0.965 mol ethanol/g of equivalent glucose consumed with ethanol titer of 10.359 g/L and hydrogen yield significantly increased to 0.857 mol hydrogen/mol of equivalent glucose consumed with hydrogen titer of 3.78 L/L of the fermentation medium. In terms of biomass, the ethanol yield was 0.0953 g ethanol and 34.78 mL hydrogen/g biomass utilized. Effect of substrate addition on batch fermentation involving FBB disclosed improved ethanol and hydrogen titer. Overall substrate consumption increased by 25.45 % for batch fermentation involving FBB. Ethanol yield of 1.034 mol/mol of equivalent glucose consumed, whereas 0.864 mol hydrogen/mol equivalent glucose consumed were found. In terms of biomass, 0.102 g ethanol and 37.928 mL hydrogen/g biomass were produced following co-fermentation by C. thermocellum DSMZ 1313. To alleviate the effect of ethanol induced inhibition, the in situ gas stripping was performed through the culture medium. The result re-confirmed the finding that glucose conversion into product could be enhanced when the inhibitory metabolite(s) were stripped from the fermentation broth. The substrate consumption improved up to 27.507 % in comparison to previous batch experimentation. Fed batch fermentation involving FBB and gas stripping produced 0.976 mol ethanol and 0.875 mol hydrogen/mol of glucose consumed. In terms of biomass, 0.096 g ethanol/g biomass with ethanol titer of 16.734 g/L and 35.09 mL hydrogen/g biomass with hydrogen titer of 6.1L/L of fermentation medium were produced at the end of fermentation. Finally, under non-aseptic conditions, fed batch fermentation involving FBB yielded 0.857 mol ethanol/mol of equivalent glucose consumed and 0.957 mol hydrogen/mol of equivalent glucose consumed. The comparison of aseptic and non-aseptic conditions revealed that ethanol fermentation decreased by 16.07 % whereas hydrogen fermentation increased up to 14.7 % in comparison with ethanol and hydrogen productions under aseptic conditions. In terms of biomass 0.084 g ethanol and 45.136 mL of hydrogen/g of biomass were produced. Principal aim of this study was to extract the energy from renewable waste sugarcane bagasse under non-aseptic extreme conditions in the form of bioethanol and biohydrogen. Successful exploitation of low cost substrate for biofuels’ production under moderate alkali/thermophilic conditions appeared promising for development of large scale bio-fermentation processes. It is foreseeable that understanding of non-aseptic extremophilic fermentations utilizing agro-industrial wastes as necessitated in the present investigation, for development of large scale cost-effective, eco-friendly biofuels generation processes will pave the way to achieve one of the greatest benefits of mankind." xml:lang="en_US