## 3 Results and Discussion
### Amplicon Metagenomic Sequencing After Storage of BSG and SCG
Compared to the aerobic conditions present in trays, under anaerobic conditions, the BSG stored in drums showed a more variable yet distinctively fermentative bacterial microbiome. Bacilli are present in all samples and are especially prevalent in the bottom samples. This is surprising as bacilli are typically aerobic, and the drum bottom is expected to have an anaerobic environment. This may be due to _B. coagulans_, the most prevalent _Bacillus_ species, having been reclassified as the facultative anaer _Heymdrickxia coagulans_, originally isolated from spoiled canned milk products [61]. The middle sample from drum 1 is dominated by the acetic acid bacterium (AAB) _A. lovaniensis_, while drum 2 has a high proportion of LAB, specifically _Limosilactobacillus fermentum_ (formerly known as _Lactobacillus fermentum_). The middle of drum 2 instead has a significant presence of _Lachnoclstidium_, a proposed genus in the class of Clostridia, described as obligate anaerobes producing acetate as major end-products from mono- and disaccharides [62]. The difference in microbiomes is believed to be due to differences in oxygen prevalence--AABs require the presence of oxygen to metabolize their substrates, while Clostridia lack aerobic respiration and do not tolerate oxygen. The microbiome dynamic observed in drum 1 is a metabolic synergy between LAB and AAB. LAB produce lactic acid either by a homofermentative glycolytic pathway, or a heterofermentative phosphoketolase pathway. The genus _Lactobacillus_, after the taxonomic revision, now contains exclusively homoformentative species producing lactic acid as the major product from hexoses [40]. The genus _Limosilactobacillus_ was split from _Lactobacillus_ and contains only heterofermative species, such as _L. fermentum_, producing lactic acid, CO\({}_{2}\), and acetate or ethanol as major products [40]. AAB are named for their ability to convert ethanol to acetic acid, while _Acetobacter_ are distinguished by being able to oxidize lactate and acetate into CO\({}_{2}\) and H\({}_{2}\)O [63]. AAB such as _A. lovaniensis_ are therefore able to metabolize the end-products from LAB such as _L. fermentum_. Therefore, LAB and AAB jointly proliferate under microexerobic conditions, as evidently present in drum 1. In addition to the metagenomic results reported here, a taxonomic heatmap for the top 35 bacterial species identified can also be found in Figure S11 in the Supplementary Materials. With respect to coffee grounds, the microbiome of SCG-2 used in tray storage and SCG-3 used in drum storage, both from the recycling facility, contain similar genera, but in different proportions. Figure 4 shows the top 10 genera and species of bacteria and fungi identified in SCG by amplicon sequencing. SCG-2 has over 50% relative abundance of _Limosilactobacillus_, with _L. panis_ being the major identified species, while the SCG-3 microbiome is dominated by _Lactiplantiobacillus_. _Acetobacter_ is more prevalent in SCG-2, and _Ligilatchocillus_ (_L. acidipiscis_) is more prevalent in SCG-3 but is present in both. Genera such as _Lactobacillus_, _Bacillus_ (_B. coagulans_), _Pseudomonas_, _Liquorilactobacillus_ (_L. nagelli_ and _L. viin_), _Lacticasibacillus_ (_L. camelliae_), _Enterobacter_, and others have minor abundance in the SCG as received. The fungal microbiomes in Figure 4b,d similarly show the same genera, but with differences in their prevalence. SCG-2 is dominated by _Pichia_ (_P. kudriavzevii_ and _P. manshurica_), with about 50% abundance, while _Candidia_ (_C. orthopsilosis_) is prevalent in SCG-3, with about 35% abundance. A number of filamentous fungi from the genus _Aspergillus_, including _A. flavus_, _A. welwithschiae_, and _A. piperis_, were identified. Note that SCG-1 was not analyzed by metagenomic sequencing due to difficulties in extracting high-quality DNA, likely due to the low TPC. SCG storage in trays led to a proliferation of _Acetobacter_ at over 60% relative abundance in all samples, followed by _Ligilatchocillus_ (_L. acidipiscis_) and _Bacillus_ (_B.