Global environmental concerns connected to the use of fossil fuels have forced the development ofalternative sustainable energy technologies. The application of anaerobic digestion, from waste streams thatcurrently have no use, can be utilized for bioenergy production. Due to the high protein and fat content,slaughterhouse waste has a high potential for biogas production. However, potential inhibitory compoundscan be formed during the degradation of the proteins and lipids, which can make the process sensitive andprone to failure. One of the ways to overcome these problems is co-digestion with carbohydrate-rich cosubstratesi.e., a mixture of agro-wastes with low protein/lipid content. This also leads to a better nutritionalbalance and enhanced methane yield due to the positive mixture interactions.

In this study, four different waste fractions, i.e., solid cattle slaughterhouse waste (SB), manure (M),various crops (VC), and the organic fraction of municipal solid waste (MSW) were investigated in monodigestionand co-digestion processes. Different mixture ratios were prepared, and the methane yield (YCH4),the specific methanogenic activity (SMA), and a kinetic parameter (k0) were determined using the batchdigestion assays at thermophilic conditions (55oC). The SB had a lower degradation rate and lower SMAcompared with those of the other samples. In order to investigate the effect of the temperature, a selectedmixture ratio was also digested at mesophilic conditions (37oC), which resulted in a decrease in YCH4 and inthe kinetic parameters, specific methane production rate (rsCH4), and k0, by up to 57% compared to thoseobtained at the thermophilic conditions. During the next part of the work, a four-factor mixture design wasapplied aiming to obtain possible synergetic or antagonistic effects. The performance of the process wasassessed using YCH4and rsCH4as the response variables. Mixing all four of the substrates resulted in a 31%increase in the YCH4compared to the expected yield calculated on the basis of the methane potential of theindividual fractions and 97% of the theoretical methane yield, clearly demonstrating a synergistic effect.Nevertheless, antagonistic interactions were also observed for certain mixtures. In order to maximize boththe response variables simultaneously, a response surface method was employed to find the optimalcombination for the substrate mixture.

The impact of the mixture interactions, obtained in the batch operation mode, was also evaluated undersemi-continuous co-digestion. Digestion of the SB as the sole substrate failed at an organic loading rate of0.9 gVS L-1d-1, while stable performance with higher loadings was observed for mixtures that displayedsynergy earlier during the batch experiments. The combination that showed the antagonistic effects resultedin unstable operation and poor representation of methanogens. It was proved that synergetic or antagonisticeffects observed in the batch mode could be correlated to the process performance, as well as to thedevelopment of the microbial community structure during the semi-continuous operation.

In the last part of the work, the response of the methanogenic biomass to the consecutive feeding applied inthe batch assays was evaluated regarding process parameters such as YCH4, SMA, and degradation kinetics.The objective was to examine whether there is a possibility to correlate these findings to the expectedprocess performance during the long-term operation. Digestion of the SB alone showed a total inhibitionafter the second feeding, which is in correlation with the failure observed during the semi-continuous mode.Furthermore, enhanced SMA was observed after the second feeding in those mixtures that showed synergyin the previous batch assays as well as a good process performance during the semi-continuous operation.