# Natural Materials as Carriers of Microbial Consortium for Bioaugmentation of Anaerobic Digesters
## 2 Materials and Methods
### Scanning Electron Microscopy Analysis (SEM)  
To perform the SEM analysis, after the incubation and rinsing with sterile saline solution, several particles of each carrier with immobilized bacteria were transferred to sterile vial containing 2% paraformaldehyde solution and kept in a refrigerator at 4 \({}^{\circ}\)C/24 h for cell fixation. Next, the samples were washed with sterile saline and then dehydrated using ethanol in a stepwise manner: 2 min in 30% ethanol, 2 min in 50% ethanol, 5 min in 70% ethanol, 5 min in 96% ethanol, and twice in 100% ethanol for 5 min. After dehydration, the samples were dried for 30 min at 50 \({}^{\circ}\)C in dry oven (Instrumentaria, Zagreb, Croatia). For imaging, a single bioparticle was coated with a plasma of gold and palladium for 180 s and imaged by a TESCAN Vega3 EasyProbe SEM (TESCAN Group, Brno, Czech Republic) at an electron beam energy of 7 keV.  
Identification, Antibiotic Susceptibility Testing, and Biochemical Characterization of Bacterial Species in the Microbial Consortium  
The consortium suspension was diluted and inoculated onto LB agar plates. On certain plates, single colonies were visible, and several types of morphologically different colonies were distinguishable (Figure 1). By using this method, it is not possible to claim that these were the only bacterial species in the consortium, but it is safe to presume that these bacterial species were dominant in the consortium, since they remained in notable numbers afterserial dilution. All the colonies that were clearly morphologically different were isolated in pure culture and subsequently identified with the MALDI-TOF mass spectrometry method (software version 3.0, Microflex LT, Bruker Daltonics, Billerica, MA, USA).  
By counting the colonies on comparable plates, the share of each identified species in the consortium was determined and compared between the consortium suspension, the control group and the bacteria immobilized on the carriers (Figure 1).  
All the experiments were done in triplicate and statistically compared by Student's t-test with the significance margin set at \(p=0.05\).  
Biochemical determination, including catalase and oxidase tests, was conducted for each bacterial species. For the catalase test, biomass was suspended in 3% H\({}_{2}\)O\({}_{2}\) on a microscope slide, and the appearance of oxygen bubbles was considered a positive result. The oxidase test was conducted by spreading biomass on strips (Microbiologie Bacident(r) Oxydase, Sigma-Aldrich Canada Co., Oakville, ON, Canada) and the appearance of a purple color was considered a positive result.  
Susceptibility testing of _E. cloace, E. asburiae, K. pneumoniae,_ and _L. adcarboxylata_ to imipenem was determined by a EUCAST standard disc diffusion assay [11]. The bacterial suspensions (0.5 McFarland units/10\({}^{8}\) CFU mL\({}^{-1}\)) were spread evenly over Mueller-Hinton Agar plates (MH) using a sterile swab. A disc of 10 ug imipenem (Liofilchem(r), Roseto degli Abruzzi, Italy) was placed on the surface of the agar and plates were incubated for 24 h at 37 \({}^{\circ}\)C. After the incubation, antibiotic cut-off points were determined following the EUCAST protocol.