## 4 Discussion  
Based on the results showing a high affinity for cell immobilization, the perlite proved to be a promising biocarrier for bacterial cells to augment biogas reactors. Perlite, also referred to as expanded perlite, is a naturally occurring aluminosilicate glassy volcanic rock. When this volcanic glass is heated to 760-1200 \({}^{\circ}\)C, it expands due to the evaporation of 2-5% of contained constitutive water, increasing the volume to 10-20 times its original volume. The obtained product is commercially called \"perlite\". It is a lightweight, frothy material with low bulk density, low thermal conductivity, high sound adsorption, and is fire resistant [16]. Due to such properties, perlite is widely used in the construction industry as an additive in insulation boards, concrete, or plaster. As perlite is a non-toxic and relatively cheap natural material, it was proposed as a biocarrier in a number of studies showing that microorganisms attach to perlite in high numbers and remain metabolically active. To mention a few, [PERSON] et al. [16] used perlite as a carrier of phosphate-accumulating bacteria _Acinetobacter junii_ intended to augment the tertiary phase of the wastewater treatment process. [PERSON] et al. [17] immobilized the yeast _Saccharomyces cerevisiae_ on perlite beads and improved the removal of aflatoxin M1 from milk. [PERSON] et al. [18] used perlite as a carrier of microbial consortium for oil degradation, and [PERSON] et al. [19] successfully introduced several bacterial strains immobilized on perlite into soil. In our experiment, the highest number of immobilized cells was obtained in systems with aeration, but in the system without aeration, the number of immobilized cells was also high (\(\sim\)10\({}^{10}\) cells per gram). Technologically, the perlite particles floated on the water surface and were floating during incubation in the aeration system, so we assumed this could decrease the degree of immobilization. In the second system, the particles were tightly sealed in the vial and rotated during the incubation, thus maximizing the water  
Figure 9: Cumulative yield of produced biogas after 24 days in different bioreactors as listed in Table 1 (N = 3). * Values that do not differ significantly (\(p<0.05\)) are circled. media/particle surface contact. It turned out that aeration significantly boosted the immobilization regardless of the reduced particle/water media contact. Two setups were evaluated, as they allow the cultivation of different bacteria types. With aeration, the attachment of facultative anaerobes (the obligate aerobes were eliminated during the anaerobic first step of isolation described in Section 2.3 and Figure 1) was propagated, and without aeration, the enrichment with obligate anaerobes is possible. As both bioparticle preparation systems give good results, in future research, the type of microorganism can be chosen, immobilized, and introduced into the biogas reactors. The biocarriers have been described to be used in biogas production, although not with precolonized microorganisms as in this study. [PERSON] et al. [12] introduced zeolite particles to anaerobic bioreactors producing biogas from grass silage. The SEM and metagenomics analysis showed preferential attachment of certain types of bacteria and methanogenic archaea onto zeolite during the 84 days of incubation. The immobilized cells were viable and enzymatically active, but the effect of biocarrier addition on biogas production was not reported. On the other hand, [PERSON] et al. [15] added kraft lignin and organic substrate to biogas-producing batch reactors and reported an increased cumulative yield of biogas, process stability, and intensified bacterial proliferation of inoculum sludge. In the case of this study, the addition of biocarrier itself did not affect biogas yield from the specific substrate, but the addition of bioparticles (perlite with immobilized microorganisms) and the suspension of bacterial consortium did.