The biological treatment of mine drainage (MD) using sulfate-reducing bacteria (SRB) is a technology in growing exploitation. The use of by-products as sources of electrons can make this treatment more environmentally and economically advantageous. However, the high chemical oxygen demand (COD) and the presence of recalcitrant molecules can lead to the accumulation of metabolic intermediates that acidify the system, thus interrupting the treatment. Besides, the adaptation of the inoculum to the establishment of sulfidogenesis with MD and by-product may be slow. This study aimed to investigate prompt adaptation and operation strategies that do not require additives to enable the sulfidogenic process to occur while maintaining a pH close to neutrality. The sources of electrons tested were trub (brewery residue) and crude glycerol – CG (residue from the biodiesel production). The inoculum from a methanogenic reactor was stored with a real MD for a month. The adapted inoculum was applied in a batch reactor for 168 h of hydraulic detention time, and promoted 75.8 ± 4.3% of sulfate removal from an MD with 3756.4 ± 258 mg.L−1 of sulfate using CG in a COD/SO42− ratio of 3 ratio. With higher initial substrate concentrations, acidification occurred and the treatment was interrupted. Using trub instead of CG, the acidification occurred at a COD/SO42− ratio of 3. Acidification was prevented and the best efficiencies in sulfate removal were obtained when the amount of substrate corresponding to COD/SO42− ratio of 3 was fractioned into equal parts and added over six days in the CG reactor. It was achieved 94.15 ± 1.76% of sulfate removal. With trub, the same procedure in which this COD was divided into seven parts, and resulted in a sulfate removal of 88.49 ± 1.02%. The removal of metals and metalloids were greater than 94.5% in all the systems in which the substrate supply was made fractionally, and the effluent generated presented alkalinity between 3370 and 4242 mg CaCO3.L−1, and pH between 6.8 and 7. The method of adaptation and operation applied allowed the realization of a MD treatment with quick establishment of sulfidogenesis and without the use of neutralizing additives. Finally, the effluent presented characteristics considered favorable for a later stage of post-treatment of the effluent with methane generation.