## 3 Results  
### Analysis of Perlite Bioparticles  
The procedure for isolating bacteria targeted for degradation of OC or PB biomass yielded morphologically different bacterial colonies grown on solid minimal media. In the presented research, all of the biomass from the surface of agar plates was scraped, a suspension in sterile saline (0.3%) was made, and named consortium, one for OC and PB, respectively. As this study was a preliminary \"proof of concept\", bacterial species in the consortium were not identified. Still, identification either by sequencing or by MALDI-TOF technology is planned for future research.  
The bacteria of the consortium showed great affinity for immobilization onto perlite particles, resulting in numbers as high as 2.1 \(\pm\) 0.9 \(\times\) 10\({}^{11}\) CFU per gram of perlite in aerated systems (Figure 3). The numbers of immobilized bacteria in systems without aeration were significantly less (_p_ < 0.05), 7.1 \(\pm\) 0.3 \(\times\) 10\({}^{9}\) CFU g\({}^{-1}\) for PB consortium and 3.4 \(\pm\) 0.6 \(\times\) 10\({}^{10}\) CFU g\({}^{-1}\) for OC consortium. However, both aerated and non-aerated systems formed perlite bioparticles with high numbers of immobilized bacteria. The high affinity of perlite for bacterial immobilization is due to its high porosity (Figure 4), resulting in a large surface area available for attachment, as pores are large enough to allow the cells to migrate inside and occupy the maximally available surface for biofilm formation.  
Figure 3: The numbers of bacteria immobilized onto perlite in the setup with aeration and OC (OC, AER), aeration and PB (PB AER), or in anaerobic conditions with mixing and OC (OC AN) and PB (PB AN), respectively. * the values that do not differ significantly (_p_ < 0.05) are circled.  
Figure 2: Anaerobic digestion experimental setup.  
From the SEM analysis, we can conclude that the bacteria attached to the smooth surface of perlite particles occupy the macro and micropores (Figures 5-8). The bacteria formed a mix-communities biofilm, judging from distinguishable different bacterial shapes and visible traces of the exopolymeric substances (EPS) (Figure 5). It should be noted here that the dehydrating procedure using alcohol destroys the EPS, which was probably present in a larger amount before imaging. The bacteria are still nicely visible due to the rigid cell wall. From obtained images, we can qualitatively compare different bioparticle preparation setups and estimate that in the case of aeration and PB consortium, there was the highest number of immobilized bacteria, which were almost uniformly dispersed all over the perlite surface (Figure 6). Similarly, many bacteria were immobilized in the case of aerated OC consortium, but in this case, in larger clusters (Figure 5). In the case of both systems without aeration, for OC and PB, a lesser amount of mostly dispersed bacteria was observable (Figures 7 and 8). Additionally, the shapes of the bacteria that could be seen were mostly different from aerated systems. It must be disclaimed here that the SEM interpretations are qualitative, and conclusions are drawn from a single randomly picked bioparticle. The observations are nevertheless in high concert with the numbers of bacteria resulting from quantitative analysis.  
Figure 8: SEM image of bacteria immobilized on perlite in an experimental setup without aeration (anaerobic) and with Pruned Biomass (PB) consortium.  
Figure 6: SEM image of bacteria immobilized on perlite in an experimental setup with aeration and Pruned Biomass (PB) consortium.  
Figure 7: SEM image of bacteria immobilized on perlite in an experimental setup without aeration (anaerobic) and with Olive Cake (OC) consortium.