## 3 Results and Discussion
### 4-n-NP Mineralization by Bacillus safensis CN12  
Based on the gene expression results of _mhqO_, there are strong indications that _B. safensis_ CN12 could be a 4-n-NP-mineralizing bacterium since this gene encodes an enzyme that breaks the aromatic ring of some compounds known to be TPs of 4-n-NP. To explore the possibility of 4-n-NP mineralization by _B. safensis_ CN12, mineralization tests in a solution were conducted, although no mineralization was observed. The main reason to explain this fact could be the lack of specific enzymes for certain intermediate compounds generated. Although a bacterium may possess genes encoding certain enzymes that break the aromatic ring, it may lack other enzymes necessary to fully degrade the resulting intermediate products [70]. Mineralization tests using _B. safensis_ CN12 were also carried out when 4-n-NP was previously adsorbed on sewage sludge to observe if the presence of other specific enzymes provided by the endogenous microbial community in sludge could help to obtain its mineralization. Several tests were carried out, and the results are shown in Figure 4. When 4-n-NP was added to sewage sludge without any other treatment or bacterial inoculation (Sludge treatment), only a low mineralization rate of about 7% was observed due to the activity of its endogenous microbial community. However, our previous studies carried out under the same conditions [36] showed a high percentage of NP degradation (>80%) after 40 days of incubation, but the present study shows that, nevertheless, mineralization does not occur. This indicates that the sludge endogenous microbiota was able to degrade adsorbed 4-n-NP, generating various TPs. However, it was only capable of mineralizing a very small fraction of these TPs into CO2. It is important to highlight that some of these non-mineralized TPs are even more toxic than the parent compound, thereby increasing the overall toxicity of sewage sludge. Inoculation with _B. safensis_ CN12 (sludge + _B. safensis_ treatment) produced a similar low mineralization rate (4.2%) (Figure 4). It is probably due to the great tendency to remain adsorbed on the hydrophobic surface of sludge organic matter that presents not only 4-n-NP not degraded by the sludge endogenous microbiota (with log Kow 5.76) but also its TPs, which maintain their aromatic moiety due to their high hydrophobic character. To overcome recalcitrant adsorption onto sewage sludge, cyclodext have been previously used as availability enhancers for a wide variety of hydrophobic contaminants [71; 72; 73], as well as for 4-n-NP [74; 75], to improve their extraction from different matrices, facilitating their degradation by the strains selected for each contaminant. Moreover, CDs exhibit minimal adsorption onto environmental matrices, demonstrate very low toxicity to microbial populations, and possess high biodegradability due to their composition of glucose units, which significantly reduces their residues in the environment [37]. The previous study by [PERSON] et al. [36] showed that the use of hydroxypropyl-\(\beta\)-cyclodextrintrim (HPBCD) as an availability enhancer allowed for the extraction of 4-n-NP strongly adsorbed on sewage sludge, facilitating its complete degradation by the endogenous microbiota in only 30 days. But, despite this, its mineralization is very low, as shown in Figure 4 (only 9.9% after almost 4 months, sludge + HPBCD treatment), indicating that only the indigenous microbiota present in sewage sludge was not able to mineralize 4-n-NP even if it was made bioavailable using HPBCD to extract it from sludge. On the contrary, when _B. safensis_ CN12 was inoculated in the presence of HPBCD, an increased mineralization rate of >33% was achieved (Figure 4, sludge + HPBCD + _B. safensis_ treatment), demonstrating the suitability of HPBCD as an availability enhancer to facilitate not only the degradation of 4-n-NP but also its mineralization, but only if _B.