A practical source of mixed cultures can easily be obtained from industrial wastewater treatment plants since they are non-sterile. However, this provides a very complex microbial community which includes together hydrogen producing and hydrogen consuming bacteria. Therefore, it is important to select a proper pretreatment method that will adequately select for the hydrogen producers and reduce or eliminate the hydrogen consumers. It is not usually possible to produce hydrogen by using typical anaerobic sludge because in anaerobic digesters hydrogen serves merely as a transitory intermediate, being almost immediately consumed in a CH4 producing reaction. By using the physiological differences between the cultures using pretreatment can eliminate competing organisms from the system. The successful application of various pretreatment methods; heat shock, chemical, acid, alkaline, ultrasonic, etc. has been reported. Each method has its own efficiency according to its composition. Combinations of different methods can also be used. Therefore, since the mixed culture does not have the same nature before using it for dark fermentative hydrogen production it is important to select the best suitable pre-treatment according to process efficiency and economical considerations [87].