Biogas is produced during anaerobic degradation of organic material and mainly consist of methane and carbon dioxide. During the anaerobic degradation, the products are fatty acids such as propionate, butyrate and acetate. In the biogas process a syntrophic cooperation between different microorganisms are important, this involves syntrophic acetate oxidizing bacteria (SAOB) which degrade acetate, syntrophic propionate oxidizing bacteria (SPOB) which degrade propionate and methanogens which produces methane. The propionate degradation is often the slowest step in the biogas process since the microorganism involved do not get much energy from it. It has been observed that FeSO4 and FeCl2 improved the propionate degradation rate in thermophilic SPOB enrichment cultures but no impact on mesophilic enrichment cultures was observed.  The aim of this study was to determine the impact by Fe/S-addition on the key species (SAOB, SPOB and methanogens) involved in the propionate degradation in mesophilic enrichment cultures and cultures with sludge with high ammonia concentration. Furthermore, cultivation studies are performed to reveal the optimal concentrations of iron (FeCl2), which boost the propionate degradation rate in mesophilic SPOB-cultures.  The cultivation study was performed in batches (<500 mL) with 50 mM propionate, 0.3 mM ammonium and different FeCl2 concentrations between 0 to 20 mM. To determine the optimal FeCl2 concentration, the cultures were analysed by measuring the propionate, methane and acetate concentration using High Performance Liquid Chromatography (HPLC). The mesophilic enrichment cultures were treated with FeSO4, FeCl2, NaSO4, FeS, FeCl3, Fe2(SO4)3 and Na2S. The mesophilic cultures with sludge were treated with FeSO4, FeCl2, FeCl3, and a combination of all three compounds. The impact of Fe/S compounds on SAOB, SPOB and methanogens were analysed on the mesophilic enrichment cultures and cultures with sludge by measuring the copies of 16S rRNA gene using quantitative PCR (qPCR). It could be concluded that in the mesophilic enrichment culture none of the Fe/S compounds affected the SPOB or methanogens, but FeS increased the concentration of SAOB. In the mesophilic culture with sludge, FeCl2 and FeCl3 increased the SPOB level, however, FeSO4 decreased the SPOB level. Moreover, the SAOB levels decreased for FeCl3 and when all three compounds were combined. The methanogen level decreased over time by the compounds FeCl2 and FeSO4. From the cultivation study, the optimal FeCl2 concentration was 20 mM.