# Natural Materials as Carriers of Microbial Consortium for Bioaugmentation of Anaerobic Digesters
## 4 Discussion  
Based on the results, all materials proved to be promising biocarriers for the microbial consortium intended to be used in biogas production. Perlite, an aluminosilicate material with high porosity, previously demonstrated great potential as a biocarrier for immobilization of phosphate-accumulating bacteria _Acinetobacter junii_, resulting in a high number of immobilized bacteria (12.65 \(\times\) 10\({}^{9}\) CFU g\({}^{-1}\)) [12]. Its efficiency in augmenting biogas-producing reactors using olive waste as a substrate was also reported. A high number of immobilized bacteria on perlite suggests its suitability for various applications in biogas production [3]. There was no statistical difference in the number of immobilized  
Figure 5: Share of bacterial species in consortium suspension, control group, and after immobilization on each carrier. The share of _E. indicium_ in the consortium suspension was 14%, whereas in the control group and after immobilization it was less than 1%. Figure 6: Number of _E. inidcium_ immobilized on NZ, ZeoSand®, perlite, and corncob. Mean values and standard deviations from triplicate experiments are shown. There was no statistically significant difference between the samples (\(p>0.05\)). bacteria between the NZ and ZeoSand(r). The most obvious difference between the two zeolite samples is in the fraction of clinoptilolite mineral, 50-55% in NZ and >80% in ZeoSand(r), suggesting that clinoptilolite content in the zeolitized tuff does not influence the immobilization of bacteria, already shown for other NZ samples [10]. On NZ, a great rate of immobilization was reported for bacteria such as _A. junii_ (1.27 \(\times\) 10\({}^{10}\) CFU g\({}^{-1}\)), _Escherichia coli_ (4.53 \(\times\) 10\({}^{8}\) CFU g\({}^{-1}\)), and _Enterococcus faecalis_ (1.3 \(\times\) 10\({}^{9}\) CFU g\({}^{-1}\)) [9]. Immobilization of microorganisms on NZ has proven to be beneficial for biogas production. In the anaerobic digestion of grass silage, bacteria immobilized onto NZ enhanced the enzymatic activity of xylanase, which is responsible for the degradation of xylan, a cell wall component in grass biomass [13]. Corncob is a material mostly made of lignin, cellulose, and hemicellulose. It has a high bulk density and provides a large amount of biodegradable carbon to immobilized bacteria [14]. Moreover, SEM visualization revealed a perforated structure with outgrowths that increased the surface area available for the attachment of bacteria. Corncob was shown to be suitable carrier for ureolytic bacteria due to its wrinkled and porous structure that allows bacteria to immobilize and survive [14]. However, it was confirmed that corncob is not an appropriate carrier for plant growth-promoting rhizobacteria due to high carbon content and poor adherence [15]. Furthermore, corncob was not ideal for enriching anammox bacteria, but it could be used in research on denitriying bacteria [16]. In general, the success of bacterial immobilization onto carriers is affected by several physical and chemical properties of the materials, mostly the particle size, as it has been observed that an increase in particle size leads to a decrease in the number of immobilized bacteria [7]. In further research with materials used here, the particle size of NZ and ZeoSand(r) could be reduced to a size of <0.125 mm, which has proven effective in achieving a higher number of immobilized bacteria [10]. In SEM images of perlite, aside from the outer surface, bacteria are visible in the micropores, which is consistent with the fact that the porous structure of perlite allows greater efficiency in the immobilization of bacteria [3]. In this research, expanded perlite was used, which is confirmed to influence the extent of bacterial immobilization compared to semi-expanded and non-expanded perlite [12]. To achieve the higher rate of immobilization, it is important for the carrier to have an irregular surface for bacterial colonization, as evidenced by many specific outgrowths on the SEM images of corncob.