The aim of this doctoral dissertation was to improve the production of methane by mechanical and enzymatic pretreatments of organic substrates. For anaerobic digestion of different substrates and determination of biomethane potential, Automatic Methane Potential Test System (AMPTS II) device was used. AMPTS II is an analytical laboratory scale device used in measurements of ultra low speed production of biomethane produced during the anaerobic digestion of biodegradable substrates. Although biogas yields of biogas plants arelimited to the specific substrates, methane yields are difficult to generalize from the collected data in published literature due to different modifications of substrates, including particle size reduction or chemical pretreatment all affecting their properties and energy recovery. Therefore, for biogas plant decision making the data need to be derived from inoculum of concrete biogas plant and substrates in such a condition as to be dosed in anaerobic reactors on real industrial scale. By upgrading commercial system AMPTS II from 0.5 liters to 5 liters, a pilot scale device was developed and validated for such real-scale experiments mirroring anaerobic reactors at the biogas plant. On 5 liter pilot scale biomethane yield of 24 substrates in solid or liquid form was assessed and compared with existing published data. By adding a variety of additives in the form of enzymes and micro-organisms in the process of anaerobic digestion, the impact on the production of methane and enhancement of the production of methane was evaluated. A combination of different substrates from paper mill sludge from Kraft process, a dehydrated paper mill sludge, bio-waste and lignocellulosic substrate an optimization of methane production in batch mode was conducted. The effects of the addition of hydrolytic enzymes on the production of methane from dehydrated paper mill sludge from Paloma was further tested in semi - continuous experiments. Significant improvements were detected. In the next following semi - continuous experiments an ultrasonic pre-treatment of sludge from the treatment plant in combination with the addition of hydrolytic enzymes was tested. In addition, the operation of the biogas plant Vučja vas was monitored for a period of 5 months during the substrate fluctuation period. Using T- RFLP technique the effects of enzyme and incubation time on the structure of bacterial and archeae consortium were assessed. By using software STATISTICA an optimal combinations of substrates in a batch mode were determined.