This two-phase, two-stage study analyzed production of biohydrogen and volatile fatty acids by acidogenic fermentation of brewery spent grains. Phase-1 served to optimize the effect of pH (4–10) on acidogenic fermentation; whereas phase-2 validated the optimized conditions by scaling up the process to 2 L, 5 L, and 10 L. Alkaline conditions (pH 9) yielded excellent cumulative H₂ production (834 mL) and volatile fatty acid recovery (8936 mg/L) in phase-1. Extended fermentation time (from 5 to 10 days) upgraded the accumulated short-chain fatty acids (C2–C4) to medium-chain fatty acids (C5–C6). Enrichment for acidogens in modified mixed culture improved fatty acid production; while their consumption by methanogens in unmodified culture led to methane formation. Increased CH₄ but decreased H₂ content enabled biohythane generation. Scaling up confirmed the role of pH and culture type in production of renewable fuels and platform molecules from brewery spent grains.