## 3 Results and Discussion
### Semi-Continuous Experiments
The fact that differences among reactors operating at the same loading, although using different substrates (either MW only or a mix between this and AS), were not detected during the initial regime phase can be attributed to two main factors: (i) the supply of nitrogen in reactors 1 and 2 (fed only with MW) was linked to reinoculation (see Table 2), operating to re-establish the process after the already mentioned overloading, and (ii) the adaptation to _d_-limonene during the start-up phase of the microbial population of reactor 3 (the one with the highest _d_-limonene loading, as reported in Table 2) was the only one not needing reinoculation. The reasons for the reduction of the methane production after about 1\(\cdot\)HRT in the regime phase in reactors 1 and 3 can be attributed to two different factors. For reactor 1, the reduction of nitrogen below tolerable limits is evident (see Figure 3f) and this supports the idea that C/N optimization is essential for stable anaerobic digestion. Indeed, adequate nitrogen presence in digesters must be ensured since it is involved in the fundamental activities of microbial metabolism (synthesis of proteins, enzymes, ribonucleic acid (RNA), and deoxyribonucleic (DNA)) [32]. Thus, the lack of nitrogen could have affected the anaerobic bacteria's metabolism, eventually causing process failure. For reactor 3, besides the nitrogen reduction also detected in this case, the feeding of the new AS (see Table 2) most probably increases the _d_-limonene concentration up to intolerable levels and triggers an inhibition of the methanogenesis, as the sharp increase in VFA concentration and in FOS/TAC and the significant pH reduction both witness. The supplementation of GAC (10 g\(\cdot\)L\({}^{-1}\)) since day 74 and until the end of the experiment causes an almost immediate recovery of the reactor, with a sharp increase in methane production since day 77 and until the end of the experiment. These results confirm previous research [15; 21] on the potential of this material in sustaining the anaerobic digestion of _d_-limonene containing substrates. Also, if the experiment, as already mentioned, was forcedly terminated after about 10 days since the beginning of the GAC addition, its effect on the process stabilization (probably mainly linked to the adsorption of _d_-limonene) are evident. It is interesting to note that in reactor 4, with a potential _d_-limonene concentration close to 450 mg\(\cdot\)L\({}^{-1}\), the process does not demonstrate signs of disruption, confirming the potential of the microbial community to adapt to _d_-limonene and therefore the importance of an optimized loading when using AS for the co-digestion with a carbonaceous substrate. This optimization should aim to slowly increase the quantity fed to the reactor to keep the _d_-limonene concentration at a tolerable level. On the other hand, two other factors are very important and worth noting: (i) optimization of the recovery of _d_-limonene during the extraction of the fish oil would be beneficial for the entire biorefinery scheme, and (ii) GAC is confirmed as a powerful additive in the anaerobic digestion of substrates containing _d_-limonene. The yield of the process was lower than expected, compared to the BMP value for the co-digestion of MW and AS (420 \(\pm\) 23 NmL\(\cdot\)gVS\({}^{-1}\)) with respect to batch tests; the reduction is evident and in the order of 40% for the low loaded reactors and 50% for the others. Furthermore, this reaction is more pronounced than the 10-30% often reported in the scientific literature for batch and semi-continuous tests on the same substrate [33; 34; 35]. Since only reactors also fed with MW display similar behaviour, this situation is most probably attributable more to the imperfect start-up of the reactors than to the use of AS a co-substrate.