Currently, dark fermentation is the most practically applicable for the implementation of biotechnological  roduction of hydrogen. However, this process has certain limiting factors, since a significant part of the substrates are converted into various metabolic products, but not into H2 . Therefore, it is necessary to develop optimal conditions for energy recovery in the form of gaseous molecular hydrogen. Various carbohydrate-containing raw materials for hydrogen production often require pretreatment before they can be used by microorganisms. Dilute acid pretreatment represents a promising way to increase biohydrogen production. However, during acid hydrolysis of carbohydrate-containing wastes, in addition to the released soluble sugars, inhibitors of enzymatic processing, such as furfural and 5-HMF, acetic and propionic acids, etc., can accumulate. In this regard, it is necessary to select the optimal conditions for the efficient production of biohydrogen. This study investigated the production of biohydrogen during the microbial fermentation of sugars in a dilute solution of a molasses-based acid hydrolyzate using Escherichia coli and a multiple mutant. The results of the experiments showed that molasses is a valuable product as a source of carbon and energy for microorganisms in the production of biohydrogen, as well as for the production of biomass for the further production of various products with high added value.