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Each of the anaerobic sludge samples was collected at the end of the methane-production stage for microbial community analysis. They were labeled as R0, R1, and R2 for proper identification. The selected sequencing primer group for the 16S pyrosequencing permitted the establishment of direct evidence for classification of the microbial communities in each of the reactors (FitzGerald et al., 2015; Luo et al., 2015). Through this comparison, the microbial communities will reveal noticeable differences in the taxonomy of the reactors effluents with and without biochar additives as it relates to hydrolysis and the methane yield.
Archaea communities; There are 15 types of Operational Taxonomic Units (OTUs) identified from the microbial archaea genus taxonomic analysis (Figure 7a). The most abundance methanogens illustrated via the genus level were the Methanothrix, while Methanobacterium and Methanomethylovorans were detected as the second most abundant in the biochar additives. These are hydrogenotrophic methanogens that can be generally found in an anoxic environment (Romesser et al., 1979) and sometimes they are found to be dominating in the anaerobic digester system (Schlüter et al., 2008). Further, methanogens abundance of methanobacterium was (0.27%) sequence read in the R0, while abundance multiplied in the R1 (1.25%) and also an increase of (0.38%) was witnessed in the R2 reactor. In this study, these results suggested that R1 reactor promotes the growth of Methanobacterium than R2 and R0. Hypothetically, it can be a reflection of R1 reactor stability performance at higher organic loading rate that was induced by the rate of the fermentative or combined acidogenic activities. Since the methanogenic abundance of Methanobacterium was only investigated at stable OLR of 6.0gVS/L.d, further investigation on characterization of its functionality is encouraged in this direction. Unclassified archaea methanogens on the surface of the bio-char sludge samples accounted for about 4.99% with dominance in the inoculum sludge, thus indicating an unclear metabolic capability of the sludge. The abundance of Methanothrix in the sludge of biochar amended reactors can equally be deduced to have played significant roles in acetate conversion to methane (Uemura and Harada, 1993). The biochar might also had promoted the interspecies mass transfer and electron transfer for methanogens performances.
Methanosarcina population in both R1 than R2 almost accounted for 0.5%, and it resides within the core zones of the biochars. (Kaparaju et al., 2008) reported Methanosarcina significantly assist in the roles for the establishment of methanogenic regions in the substrates during the operations of anaerobic digestion. However, it is noteworthy in this study, that Methanothrix which is an obligate acetoclast (FitzGerald et al., 2015) was much abundance. It probable out-strived Methanosarcina by its higher growth rate and tolerance against inhibition on long run operations.
For the bacterial taxonomy, a total of 50 types of OTUs was detected showing diversities of bacterial microbes (Figure 7b). Interestedly, it was detected that the bacterial community varieties were more than triple as compared with the archaeal. However, with relative consideration to the role of the methanogens components, they cannot be compared with the archaeal communities. Bacteria microbes that were detected and widely known to be active for hydrolysis and carbohydrate fermentations are the (Lachnospiraceae, Peptostreptococcaceae, Ruminococcaceae and Gelria, Christensenellaceae : Sedimentibacter Tissierella, Syntro- phomondaceae), these are dominance in R1 and R2 reactors. The second most abundant genus is the Bacteroidetes, these are commonly recognized in the anaerobic environment and principally responsible as fermenters and acidogens. Proteobacteria, Rhodobacteraceae, Granulosiococcaceae,Nannocystineae;Desulfomicrobium;Alteromonadaceae, Nannocystinaceae, Granulosiococcaceae among others existed in R0, R1 and R2. These are extensively scattered and varied constituents of fermenters commonly found in the anaerobic digestion.

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