## 3 Results and Discussion
### Amplicon Metagenomic Sequencing After Storage of BSG and SCG  
In order to characterize the microbiome, bacterial 16S rRNA and fungal ITS amplicon metagenomics sequencing was conducted on the solid sidestreams as received and after 14 days of storage. The metagenomic sequencing results at the genus level are summarized for BSG in Figure 3, showing the top 10 genera and species of bacteria and fungi identified by amplicon sequencing. BSG-1, from the craft brewery, has a very different bacterial metagenomic sequencing result from BSG-2 and BSG-3 from the recycling facilities. The dominant results from BSG-1 are _chloroplast_ and _mitochondria_, arising from the BSG material itself and indicating a very low microbial load as also found by TPC. In contrast, significant bacterial loads were found by TPC for BSG-2 and BSG-3, and the metagenomic sequencing found _ Bacillus_ (_B. coagulans, B. smithii_, and _B. thermoamylovorans_) to be the dominant genus. _P.enibacillus_ (_P. tumbe, P. cookie_, and _P. barengoltzii_), _Lactobacillus_ (_L. amyloticus_ and _L. delbrueckii_), and _Acetobacter_ (_A. lovaniensis_) are also prominent in BSG-3, while other genera have minor  
Figure 2: Total plate count (TPC) on wet matter basis of BSG and SCG before and after 14 days of storage in trays (BSG-2) and drums (BSG-2 and SCG-3): (**a**) log\({}_{10}\) TPC of bacteria in BSG; (**b**) log\({}_{10}\) TPC of fungi in BSG, (**c**) log\({}_{10}\) TPC of bacteria in SCG; (**d**) log\({}_{10}\) TPC of fungi in SCG. contributions to the BSG microbiome. The fungal microbiome characterization of as-received BSG was inconclusive, with only BSG-3 having _Kluyveromyces_ (_K. marxianus_) and _Pichia_ (_P. kudriuzevii_) identified at the species level, as seen in Figure 3a,c. These results generally agree with those obtained by microbial isolation and identification and shown in Table 4, but with some differences in the assigned genus and species identified. Storage of BSG in trays substantially altered the microbiome, with the dominance of _Bacillus_ bacteria being supplanted by _Enterococcus_, _Weisella_, and _Paenibobacillus_, with some differences between the two trays and sampling positions, especially for the species identified, as per Figure 3. The fungal microbiome on storage in trays is dominated by filamentous fungi of the genus _Aspergillus_ (_A. flavus_ and _A. fumigatus_), with over 50% relative abundance in all tray samples. Aspergillus, especially _A. flavus_, are known spoilage microorganisms in BSG, known to potentially produce highly toxic compounds known as aflat [59]. This taxon was dominant in all tray samples on storage as shown in Figure 3b. The other major filamentous fungus identified was _Mucor_ (_M. indicius_), which is found in a number of traditional Asian fermented foods [60]. _Alternaria_ and _Pseudogymnoascus_ genera were also found with low prevalence. _Alternaria_ spp. have previously been associated with BSG spoilage [59]. A number of yeasts were identified, including all three yeasts isolated and identified previously: _Kluyveromyces_ (_K. marxianus_), _Pichia_ (_P. kudriuzevii_, _Candida_ (_C. tropicalis_ and _C. orthopsilosis_), _Dititina_ (_D. mesorgosa_), _Cyberlindera_ (_C. fabianii_), and _Meyerozyma_ (_M. guillermondii_). In conjunction with the high bacterial and fungal TPCs for BSG under aerobic storage, it is evident that growth of  
Figure 3: Amplicon metagenomic sequencing of BSG-1, BSG-2, and BSG-3 as received and BSG-2 after 14 days of storage in trays and drums: (**a**) top 10 genera of bacteria; (**b**) top 10 genera of fungi; (**c**) top 10 species of bacteria; (**d**) top 10 species of fungi identified by 16S rRNA and ITS amplicon sequencing. Note that due to negligible fungal TPC, drum samples were not ITS-sequenced. Enterococcus (E. durans_ and _E. facecium_) and _Aspergillus_ (_A. flavus_ and _A. fumigatus_) is a key concern not just for food safety, but also for animal feed safety and quality due to aflat.