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Seaweed (or macroalgae) produced sustainably at large scale opens opportunities as source of fuels, chemicals and food. The production does not directly compete with terrestrial food production and may make use of anthropogenic sources of carbon dioxide and nitrogen. Seaweed biomass can be transformed into a suitable substrate for fermentation using a biorefinery approach. In this study the entire process of biofuel production from seaweed is described: starting with cultivation and harvest, the seaweed is dried and cut, enzymatically hydrolysed, demineralized, detoxified, and finally fermented into acetone, butanol, and ethanol (ABE). Juvenile Saccharina latissima was directly seeded on AlgaeTex® nets and cultivated in the North East Atlantic off the west coast of Scotland for 6 months. Sun dried seaweed was hydrolysed with different enzymes, looking for optimal glucose release, solid/liquid ratio, and enzyme load. Using Cellic® CTec2 in combination with alginate lyases, approximately 80% of available glucose was released. The hydrolysis was scaled up to 100 L, using only Cellic® CTec2. Part of the hydrolysate was demineralized using ion-exclusion chromatography, removing over 90% of minerals while recovering 92% of glucose and mannitol. A fraction of the demineralized hydrolysate was additionally detoxified using a hydrophobic resin to remove hydrophobic components to a concentration below detection limit. The three hydrolysates (untreated, demineralized, and demineralized followed by detoxification) were used as substrate for ABE production by a newly developed strain of Clostridium acetobutylicum adapted to grow on S. latissima hydrolysate. Demineralization reduced the lag phase of fermentation from 72 h (untreated) to 24–48 h. Further detoxification of the hydrolysate led to immediate fermentation, resulting in a yield of 0.23 ± 0.02 g ABE /g sugar similar to control fermentation in control medium (0.19 g ABE /g sugar). • Complete path from seaweed cultivation to fermentative acetone, butanol, and ethanol production • Enzymatic hydrolysis with different enzymatic cocktails • Scale-up to 100 L of enzymatic hydrolysis • Chromatographic demineralization and detoxification of hydrolysate • Fermentation of hydrolysates with adapted strains of Clostridium acetobutylicum [ABSTRACT FROM AUTHOR]