# Natural Materials as Carriers of Microbial Consortium for Bioaugmentation of Anaerobic Digesters
## 4 Discussion
Further, previous research has examined the effect of the charge, expressed as zeta potential, of zeolite and perlite on the rate of immobilization. Bacterial cells are negatively charged, and the tested materials are natural and therefore also negatively charged, but not enough to be repulsive to the bacterial cells [10; 11; 12]. Therefore, surface charge could show a particular, but not crucial, effect on the rate of immobilization. The hydrophobicity presumably also did not affect the immobilization, since it was previously shown that bacteria, which are generally naturally hydrophobic, interact with both hydrophilic and hydrophobic surfaces and successfully immobilize onto NZ [7]. In view of this research, the isolated microbial consortium should not be considered a biogas producer but a biomass degrader. In the complex anaerobic digestion system, methane production is performed by methanogenic archaea, which were not a part of the described consortium. This consortium's role was to help biodegrade complex organic biomolecules contained in MxG biomass, such as lignocellulosic and cellulose, and to provide end products that methanogens could metabolize. In this manner, it is very well known that microbial consortia, compared to single species, are more advantageous for the degradation of complex compounds [17]. Microbial consortia can be either isolated or constructed. For construction, a bottom-up approach is to determine the genomes of individual species of a potential consortium and reconstruct metabolic networks and microbial interactions. This should allow the design of microbiomes with specific properties, such as distributed pathways, modular species interactions, and resilience, that would be optimized for specific ecosystem function and stability [17]. For actual bioaugmentation on an industrial scale, the approach must be as simple as possible and ready to be performedin a basic microbiological laboratory. This was the focus of the approach described here--to isolate the targeted consortia that can be adjusted to biodegrade various types of biomass, but in a simple and reproducible way, by focusing only on the bacteria that can be cultivated with ease, repeatedly for each new experiment. Using this parameter, we ended up with a consortium of five bacterial species that gave reproducible results and were identified with high certainty. Detected species in the microbial consortium were _E. cloacae_, _K. pneumoniae_, _E. asburiae_, _L. adearboxylata_, and _E. indicum_. These bacterial species are catalase-positive and oxidase-negative, meaning they are facultative anaerobic microbes [18; 19]. Facultative anaerobic bacteria should be completely metabolically functional in the anaerobic digesters, and at the same time large amounts of biomass can be easily grown in aerobic conditions, without the use of specific anaercultivators. The individual bacterial species from the bacterial suspension did not exhibit the same affinity for a specific carrier, whereas _E. cloacae_ demonstrated an ability to be immobilized on each tested carrier in huge percentages (72-88%). The difference probably arises from either carrier affinity for _E. cloacae_ or the best adaptation of this species to competition with other species in tested conditions. In a study by [PERSON] et al. [20], a microbial consortium consisting of _Enterobacter cloacae_, _Bacillus_ sp., and _Bacillus licheniformis_ was immobilized on perlite, zeolite, silica, and vermiculite. The consortium was successfully immobilized on carriers, including _E. cloacae_, although with a lower cell loading capacity on zeolite (5.10 \(\times\) 10\({}^{6}\) CFU g\({}^{-1}\)) and perlite (5.37 \(\times\) 10\({}^{6}\) CFU g\({}^{-1}\)). The same microbial consortium immobilized on perlite improved the degradation of crude oil by 25% as compared to free-living cells [21]. The most significant proportion of _L. adearboxylata_ was on perlite (23%), which is a higher result compared to the proportion in the consortium suspension (13%).