Peat moss (Sphagnum) biomass is a promising bioresource to substitute peat in growing media with a renewable material. For sustainable production on a large scale, the productivity of Sphagnum mosses has to be increased by optimizing culture conditions. Optimization was achieved using fractional factorial design and response surface methodology based on central composite design to determine concentrations of eight factors leading to highest biomass yield. We improved a standard Sphagnum medium by reducing the concentrations of NH4NO3, KH2PO4, KCl, MgSO4, Ca(NO3)2, FeSO4 and a microelement solution up to 50 %. Together with a reduced sucrose concentration for Sphagnum fuscum, while it remained unchanged for Sphagnum palustre and Sphagnum squarrosum, moss productivities were enhanced for all tested species in shake flasks. Further upscaling to 5 L photobioreactors increased the biomass yield up to nearly 50-fold for S. fuscum, 40-fold for S. palustre and 25-fold for S. squarrosum in 24 days.