Large amounts of hydrogen are produced worldwide, which are nearly all from fossil origin. Use of biomass instead of fossil fuels to produce hydrogen can contribute to a reduction of greenhouse gas emissions. Therefore, the hydrogen has to be produced at high yield and efficiency. A Microbial Electrolysis Cell is a new technology that is able to produce hydrogen at high yield and efficiency in one step from biomass by addition of only a small amount of energy.The energy that needs to be added to produce hydrogen is determined by the internal resistance of the system, which can be divided into the different components of the system and presented as an equivalent circuit. These partial internal resistances were used to explain the difference in performance of Microbial Electrolysis Cells equipped with cation and anion exchange membranes. The better performance of the anion exchange membrane configuration was caused mainly by the much lower internal resistance due to the transport resistance of ions.The hydrogen production rate is determined by conversion rate of substrate and the coulombic efficiency of this conversion. The current density and coulombic efficiency was influenced by chancing the mass and charge transport in porous electrodes. Increase of a forced flow speed through the porous electrodes led to an increase in current density when the flow was directed away from the membrane caused by a decrease in anode resistance. Furthermore, the increase in flow speed led to an increase of the coulombic efficiency.The coulombic efficiency can also be influenced by changing the substrate concentration and the anode potential. It was shown that a higher anode potential increased the energy available for these electrogens and in that way they could outcompete the methanogens. Furthermore, also a lower substrate concentration made it possible for the electrogens to outcompete the methanogens.Finally, a system was developed that makes it possible to recycle alkalinity from waste streams in an extra recovery compartment and to produce the same current without addition of expensive buffer.In the future, improvement of the following three key characteristics can make Microbial Electrolysis Cells a competing technology for the production of hydrogen gas from biomass. Firstly, the coulombic efficiency needs to be controlled and improved to reach values close to 100% for all possible organic substrates. Secondly, the use of materials and the design should be such that the total internal resistance does not exceed values of 30 mΩ m2. Thirdly, the use of added chemicals like buffers should be limited, as the relative productivity does not increase with increasing use of chemicals.