# Natural Materials as Carriers of Microbial Consortium for Bioaugmentation of Anaerobic Digesters
## 4 Discussion
This indicates that perlite could be an appropriate carrier for _L. adearboxylata_, but information regarding the immobilization on carriers or its potential implication in bioremediation is still lacking. _K. pneumoniae_ and _E. asburiae_ could be used in the form of immobilized or free-living cells in the process of bioaugmentation. It was reported that _E. asburiae_ is a promising approach for bioremediation of organochlorine pesticides in surface water [22]. _K. pneumoniae_ could be used for the pre-treatment of food waste prior to anaerobic co-digestion with straw to decrease lipid content and enhance methane production [23]. Although the proportion of _E. indicum_ in the bacterial suspension was 14%, it was not detected after the immobilization of the consortium on any carrier. This bacterial species was present in significant amounts in the original consortium suspension but was absent after incubating the consortium in nutrient media without the carrier. However, after incubation of the pure axenic culture, it was successfully multiplied and immobilized. Therefore, the assumption is that _E. indicum_ vanishes in competition with the other four species of the consortium. One thing to consider in biotechnology is that it is important not to use potentially pathogenic bacteria due to the risk of them leaking into the environment. Although _K. pneumoniae_ was characterized as susceptible to impenem, it is well known as a member of the 'ESCAPE' pathogens, a group of clinically significant and multi-drug-resistant bacteria [24]. Instead of using potentially pathogenic bacteria, the microbial consortium can be adjusted to avoid risking public health. For further research, _K. pneumoniae_ could be removed or replaced with other bacterial species. To verify that the carriers described here could be successfully used for bioaugmentation of large-scale anaerobic digesters, the capacity for biogas yield will be tested for each carrier in subsequent research. Encouraging results have already been obtained when a bacterial consortium obtained in a similar manner was used to bioaugment reactors with olive residues as a substrate and perlite as a carrier [3]. Next, the long-term stability and performance of the carriers in the reactors need to be tested. Since all the carriers except for the crushed corncob are minerals, no degradation in the anaerobic sludge is expected. Intheory, the bioparticles would act as seeds for the added organisms for a longer period of time; however, the duration needs to be determined. When considering large-scale usage of the described biocarriers, environmental impact is very important. Therefore, the chosen carriers are completely natural and have no negative impact on the environment, making them safely disposable along with the surplus sludge. This was one of the reasons these carriers were chosen. Another reason is that all of the carriers are easily commercially obtainable and feasible, so scaling up the described technology to an industrial scale should not be a challenge. Probably the highest risk is maintaining the bacterial consortium. Therefore, all procedures were designed to be easily replicable; the consortium is kept in a Microbank and revitalized for each replicate experiment, proving its stability. The growth occurred on standard, non-fastidious nutrient media, and in aerobic conditions or tightly sealed containers, without the need for sophisticated anaerobic cultivation equipment. The five species in the consortium are only a small part of the natural diversity of the microbial consortiums in the anaerobic sludge, but according to the shown experiments, these bacterial species were targeted for the degradation of a specific substrate, in this case the _Mischuntus_ grass. If added in large enough numbers in the reactors, attached to the biocarriers, they presumably should increase the biogas yield.