This work evaluates the possibility of applying enzymatic beech wood (Fagus sylvatica) hydrolysate as a feedstock for Chlorella sorokiniana growth, and fatty acid and pigment production. Beech wood solids were pretreated with NaOH at high temperature to partially remove xylose and Klason lignin, and enable production of glucose during subsequent enzymatic hydrolysis. Neutralized wood enzymatic hydrolysate containing glucose (TGP-Enz10), was tested on Chlorella growth during heterotrophic cultivation and compared with microalgae growth in a medium containing synthetic glucose (TGP). Results show that enzymatic hydrolysate enabled Chlorella growth in the dark for biomass, fatty acid and pigment production due to the presence of glucose, although the productivity obtained was smaller, if compared to heterotrophic cultivation in a synthetic TGP medium. Partial growth inhibition and diminished productivity in wood hydrolysate supplemented Chlorella culture was due to the presence of neutralized citrate buffer. Neutralized citrate buffer (TGP-Cit10) was found to partially inhibit heterotrophic growth and also strongly suppress mixotrophic growth in Chlorella culture. This buffer was also shown to alter fatty acid composition and to slightly affect Chl Total /Car Total ratio during heterotrophic cultivation. Heterotrophic Chlorella cultivation with TGP-Enz10 showed that wood enzymatic hydrolysate can constitute a potential feedstock for microalgae cultivation, although the composition of the buffer used during enzymatic hydrolysis should be taken into consideration.