## 3 Results and Discussion
### Total Plate Count After Storage of BSG and SCG  
The microbial growth by TPC at the beginning and end of 14 days of storage was investigated for the different storage conditions. The bacteria and fungi TPCs for BSG stored in trays and drums are shown in Figure 2a,b. In trays, bacterial TPCs in the range of 9 to 10 \(\log_{10}\) CFU/g were found at the end of storage, representing a 100-fold increase compared to an initial value of 7.42 \(\pm\) 0.02 \(\log_{10}\) CFU/g. Fungal TPCs show a more striking change, with the initial value below the limit of detection (1 \(\log_{10}\) CFU/g) and a final value of 7 to 8 \(\log_{10}\) CFU/g. This suggests that aerobic storage of BSG shows substantial vulnerability to fungal contamination. In contrast, BSG in drums exhibits no detectable fungal TPC, while bacterial TPCs are similar to or slightly lower than those in trays in the middle position, but much lower than those in the bottom position with 6.12 \(\pm\) 0.27 and 6.66 \(\pm\) 0.07 \(\log_{10}\) CFU/g. Overall, these studies suggest that enclosed storage or ensilage will help to protect BSG from contamination with fungal contaminants that may give rise to mycotoxins and the degradation of feedstock quality.  
With respect to coffee grounds, the bacteria and fungi TPCs for SCG stored in drums are shown in Figure 2c,d. Bacterial TPCs were found to be in the range of 8 to 9 \(\log_{10}\) CFU/g for all positions in both replicates, representing over a 100-fold increase compared to the initial value of 6.16 \(\pm\) 0.02 \(\log_{10}\) CFU/g. Although SCG is less nutritious than BSG, high counts of bacteria are still found at the end of storage. This could be due to high surface area and porosity of SCG, which provide a favorable microenvironment for microbial colonization [58]. Indeed, even if nutrients are limited, the structure of the SCG can retain moisture, making residual nutrients present in the SCG available for microbial consumption. Conversely, fungi were only detected in the bottom sample of one of the replicates, with a TPC of 2.60 \(\pm\) 0.07 \(\log_{10}\) CFU/g, compared to 1.94 \(\pm\) 0.00 \(\log_{10}\) CFU/g fungi initially. SCG stored in drums thus appears prone to bacterial spoilage, even though reported literature on the spoilage of coffee products has focused on fungal spoilage [32; 33; 34]. A low fungal TPC of 1.94 \(\pm\) 0.00 \(\log_{10}\) CFU/g was found in the SCG-3 as received, and also in one sample from the bottom of the drum with 2.60 \(\pm\) 0.05 \(\log_{10}\) CFU/g, indicating a risk of fungal spoilage even in drum storage. These results highlight that SCG is microbially unstable towards both bacteria and fungi, with likely implications on the quality of products such as food ingredients and lipid and bioactive extracts that can be produced from SCG after industrially relevant storage.  
In addition to the TPCs reported here, bacterial and fungal abundance measured by quantitative PCR can be found in the Supplementary Materials Figure S10. These supplementary results indicate that the bacterial growth occurs rapidly, stabilizing around day 3 for BSG and day 7 for SCG, while fungal growth increases more gradually, with the exception of BSG under anaerobic storage, in which the fungal presence decreased.