The recalcitrant structures either from substrate or microbial biomass contained in digestates after anaerobic digestion (AD) highly influence digestate valorization. To properly assess the microbial biomass contribution to the digested or-ganic matter (OM), a combination of characterization methods and the use of various substrate types in anaerobic con-tinuous reactors was required. The use of totally biodegradable substrates allowed detecting soluble microbial products via fluorescence spectroscopy at emission wavelengths of 420 and 460 nm while the protein-like signature was enhanced by the whey protein. During reactors operation, a transfer of complex compounds to the dissolved OM from the particulate OM was observed through fluorescence applied on biochemical fractionation. Consequently, the fluorescence complexity index of the dissolved OM increased from 0.59-0.60 to 1.06-1.07, whereas it decreased inversely for the extractable soluble from the particulate OM from 1.16-1.19 to 0.42-0.54. Accordingly, fluorescence regional integration showed differences among reactors based on visual inspection and orthogonal partial latent struc-tures (OPLS) analysis. Similarly, the impact of the substrate type and operation time on the particulate OM was revealed by 13C nuclear magnetic resonance using OPLS, providing a good model (R2X = 0.93 and Q2 = 0.8) with a clear time-trend. A high signal resonated at similar to 30 ppm attributed to CH2-groups in the aliphatic chain of lipid-like struc-ture besides carbohydrates intensities at 60-110 ppm distinguished the reactor fed with whey protein from the other, which was mostly biomass related. Indeed, this latter displayed a higher presence of peptidoglycan (delta H/C: 1.6-2.0/ 20-25 ppm) derived from microbial biomass by 1H -13C heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance. Interestingly, the sample distribution obtained by non-metric multidimensional scaling of bacte-rial communities resembled the attained using 13C NMR properties, opening new research perspectives. Overall, this study discloses the microbial biomass contribution to digestates composition to improve the OM transformation mech-anism knowledge.