# Effect of Bioaugmentation on Biogas Yields and Kinetics in Anaerobic Digestion of Sewage Sludge
## 3 Results and Discussion
### Microbial Structure of Digesters  
Sludge fermentation starts with hydrolysis followed by acidogenesis. In both these processes, the predominant microorganisms are obligatory anaerobes and facultative bacteria. Microorganisms conducting acetogenesis produce hydrogen and are in symbiosis with methanogenic archaea, which consume hydrogen (syntrophy or interspecies hydrogen transfer--IHT). The balance between methanogens and microorganisms involved in acido- and acetogenesis is crucial because if the activity of the latter is too high, anaerobic digestion will fail due to the acidification of the reactor. The microbial structure of the biomass from the reactors operated in the first phase of the experiment was analyzed using next-generation sequencing. Rarefaction analysis was used to characterize the richness of taxa in the experimental digesters. At the genus level, the curves leveled off, indicating acceptable sampling and coverage of the richness in the samples (data not shown). The microbial diversity of the biomass was the highest in the control reactor (H\({}^{\prime}\) = 2.86) and decreased gradually to H\({}^{\prime}\) = 2.52 and H\({}^{\prime}\) = 2.16 in R2 and R3, respectively, as the dose of bioaugmenting mixture was increased. In the reactors, Bacteria predominated and Archaea constituted between 1.3% and 1.4% of identified sequences (Table 3). Figure 5: SEM micrograph of non-bioaugmented (**a**) and bioaugmented (**b**) digested sewage sludge (magnification 10\(\times\)). \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Kingdom; Phylum; Class; Order; Family; Genus** & **R1** & **R2** & **R3** \\ \hline Bacteria; Verrucomicrobiae; Verrucomicrobiae; Verrucomicrobiales; Unclassified & 3.1 & 1.0 & 0.4 \\ \hline Bacteria; Unclassified & 31.0 & 38.8 & 52.7 \\ \hline Bacteria; Thermotogae; Thermotogae; Thermotogales; Unclassified & 4.4 & 2.8 & 4.2 \\ \hline Bacteria; Synergistetes; Synergistats; Synergistates; Synergistaceae; Synergistes & 0.5 & 0.3 & 0.1 \\ \hline Bacteria; Spirochaetes; Unclassified & 2.8 & 3.9 & 3.9 \\ \hline Bacteria; Spirochaetes; Spirochaeta; Unclassified & 0.8 & 0.3 & 0.3 \\ \hline Bacteria; Proteobacteria; Unclassified & 0.4 & 0.5 & 0.3 \\ \hline Bacteria; Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Thermomonas & 0.5 & 0.4 & 0.3 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Percentage of bacterial taxa in biomass from the experimental reactors. This value was one order of magnitude lower than for example the one noted in mesophilic full-scale digesters with sewage sludge [24]; however, it was similar to the values noted in mesophilic reactors for co-digesting of fish waste and cow manure (about 1%) [7] or solid-state digesters fed with kitchen waste, pig manure and excess sludge (about 0.5%) [40]. In this study, despite being only a small fraction of the entire microbial community, Archaea ensured efficient production of methane-rich biogas. The structure of the biomass in the reactors differed from that of the mixture for bioaugmentation. This indicates that most of the microbes in the mixture were not able to survive in the reactors; however, the ones that survived improved the efficiency of methane fermentation as concluded based on the rate constant for biogas production and efficiency of SCOD removal. Within the biomass, the percentage of unclassified bacteria increased with increasing dose of bioaugmenting mixture showing that high diversity of yet unknown microorganisms was present in the reactor as a result of bioaugmentation. Among Bacteria, the core communities belonged to the phyla Thermotogae, Spirochaetes, Cloacimonetes, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, and Proteobacteria. Bacteroidetes, Chloroflexi, Firmicutes, and Proteobacteria contain most of the identified species of acidogenic bacteria that support the hydrolysis stage [41]. Microorganisms belonging to class Cytophagia predominated in the biomass (12.3-18.9%). However, as doses of the bioaugmenting mixture were increased, the percentage of _Cytoplasa_ sp.