# Natural Materials as Carriers of Microbial Consortium for Bioaugmentation of Anaerobic Digesters
## 3 Results
### SEM Analysis  
The SEM analysis revealed bacterial cells attached to the surface of the different carriers. In the ZeeSand(r) and NZ images, it was difficult to distinguish the cells clearly due to extracellular substances (Figure 3A,B). In the case of perlite, the cells are more clearly distinguishable and visible in microcolonies scattered all over the perlite surface and in the micropores (Figure 3C). The SEM analysis of crushed corncob revealed that the surface of the particles is full of outgrowths (Figure 4A), which are covered with attached cells (Figure 4B), thereby potentially increasing the surface area available for bacterial immobilization.  
Figure 3: SEM image of bacteria immobilized on ZeeSand® (**A**), NZ (**B**), and perlite (**C**). Clearly visible attached bacterial cells are yellow circled.  
Figure 2: Number of bacteria immobilized on NZ, ZeeSand®, 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).  
Share, Antimicrobial Resistance Profile and Biochemical Characteristics of Bacterial Species in Microbial Consortium  
After cultivating the consortium on agar plates that were diluted enough to have single colonies clearly separated from each other, five types of morphologically different bacterial colonies were observed. These were re-inoculated to grow pure cultures, at least three colonies of each type. All of these were identified by the MALDI-TOF MS method, and with unambiguity (MALDI score above 2.0, interpreted as High Confidence Identification), five species were identified and reasonably reliably ascribed to a specific colony type: _Enterobacter cloace, Klebsiella pneumoniae, Enterobacter asburiae, Leclercia adearboxylata,_ and _Exiguobacterium indicum_. Results revealed that in consortium suspension, the dominant species was _E. cloace,_ followed by _E. indicum_ and _L. adearboxylata,_ and in smaller share _E. asburiae_ and _K. pneumoniae._ During immobilization, the shares change in favor of _E. cloace,_ which becomes an even more dominant species with 72-88% presence, and shares for other species range from 2 to 23% (Figure 5).  
Interestingly, the share of _E. indicum_ changed from 14% in consortium suspension to <1% during immobilization, and this was the same for all the tested carriers. The share of _E. indicum_ in the control suspension was less than 1%, indicating that it was overgrown in competition with other bacteria and not because of an inability to immobilize. This was further confirmed in the experiment where a pure culture of _E. indicum_ was immobilized on NZ (\(2.18\times 10^{7}\) CFU g\({}^{-1}\)), ZeoSand(\(2.15\times 10^{8}\) CFU g\({}^{-1}\)), perlite (\(4.48\times 10^{8}\) CFU g\({}^{-1}\)), and corncob (\(2.93\times 10^{8}\) CFU g\({}^{-1}\)) (Figure 6). There was no statistically significant difference between carriers (_p_-value > 0.05).  
Based on biochemical tests, these bacterial species are catalase-positive and oxidase-negative, confirming they are facultative anaerobes. Since several of the identified species belonged to the _Enterobacterales_ group, and are potential opportunistic pathogens, the antibiotic susceptibility to carbapenem was determined. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard readings for inhibition zone diameter, _K. pneumoniae_ (24 mm), _E. asburiae_ (22 mm), and _L. adearboxylata_ (22 mm) were categorized as susceptible to imipenem. The one diameter for _E. cloace_ was 21 mm and it was described as susceptible with increased exposure.  
Figure 4: SEM image of the surface of crushed corncob particle covered with outgrowth (**A**) and bacteria immobilized on the surface of the outgrowths (**B**). Clearly visible attached bacterial cells are yellow circled.