The goal of this work was to evaluate the efficiency of two different agitation systems by measuring the nutrient distribution in a digester fed with renewable energy crops and animal manure. The study was carried out at the practical research biogas plant of Hohenheim University. A unique probe sampling system has been developed that allows probe sampling from the top of the concrete roof into different parts and heights of the digester. The samples were then analyzed in the laboratory for natural fatty acids concentrations. Three different agitation setups were chosen for evaluation at continuous stirring and feeding procedures. The results showed that the analysis approach for agitator optimization through direct measurement of the nutrients distribution in the digester is promising. The type of the agitators and the agitation regime showed significant differences on local concentrations of organic acids, which are not correlated to the dry matter content. Simultaneous measurements on electric energy consumption of the different agitator types verify that by using the slow-moving incline agitator with large propeller diameters in favor of the fast-moving submersible mixer with smaller propeller diameters, the savings potential rises up to 70% by maintaining the mixing quality.