Trace nutrients significantly affect the microbial metabolic activity within anaerobic digestion processes but always imply the risk of overdosing of heavy metals. In this study the applicability of a sequential extraction scheme established for soil and sediment samples on biogas slurries with different compositions was tested and compared to an adapted version of this extraction method. The analytical results proved the successful applicability of the developed analytical technique for the speciation of trace nutrients in anaerobic digestion systems. The procedure fulfills the basic requirements of reproducible data, a time-saving analytical approach and economic feasibility. Recovery rates of 90-110% were obtained for the most important trace elements Fe, Co, Cu, Mo, Ni and Zn. However, it was demonstrated that the adapted method provides more reliable information about the bioavailable fractions and it is considered the more appropriate approach. Data on fractionation indicated that up to 76% of these essential trace nutrients might be present in an insoluble state. Depending on the specific trace element a significant fraction, from 30% to more than 70%, is not directly bioavailable. This important aspect should be considered to guarantee sufficient supply of the microbial consortium with trace elements and at the same time to avoid overdosage. (C) 2014 Elsevier Ltd. All rights reserved.