## 3 Results and Discussion
### In Silico Analysis of Bacillus safensis CN12 Genome Sequence  
Among all NP-degrading bacterial strains isolated from sewage sludge, our _B. safensis_ CN12 strain was identified as exhibiting the highest degradation efficiency for 4-n-NP. Although no transformation products indicative of aromatic ring cleavage were detected during 4-n-NP degradation, genomic analysis of _B. safensis_ CN12 was conducted to investigate the presence of genes associated with aromatic compound metabolism. The assembled genome comprises 24 contigs with a total length of 3,673,492 bp and a GC (guanine-cytosine) content of 41.6%. The draft genome contains 3688 coding sequences, including 35 5S rRNAs, 4 16S rRNAs, 5 23S rRNAs, and 64 tRNAs. The taxonomic classification of the strain was confirmed through comparative BLAST analysis of the 16S rRNA gene, which exhibited 100% sequence identity to the 16S rRNA gene of _Bacillus safensis_ (GenBank accession: OP143733.1). The genome harbors six genes implicated in aromatic compound metabolism: three associated with quinate degradation, one with biphenyl degradation, one with gentisate degradation, and one linked to salicate and gentisate catabolism (Supplementary Figure S3). Salicylate and gentisate, key intermediates in aromatic compound degradation, are metabolized via the gentisate pathway. This pathway also participates in the catabolism of benzoates, phenolic compounds, and polycyclic aromatic hydrocarbons (PAHs) [38; 52]. Additionally, 44 genes (Figure S3), related to motility and chemotaxis were identified, which may enhance aromatic compound removal by promoting bacterial proximity to contaminants, thereby improving degradation efficiency [53]. To date, there is limited genetic information available in the literature regarding the genes involved in the degradation pathway of 4-n-NP. In general, it is known that the catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes are crucial for the ortho- and meta-cleavage pathways of aromatic rings, respectively, and play a significant role in the biodegradation of aromatic compounds [54]. Specifically, in the case of 4-n-NP, [PERSON] et al. [55] investigated the presence of the _C12O_ and _C23O_ genes in natural water microcosms from an urban river using the most-probable-number-polymerase chain reaction (MPN-PCR) method and terminal restriction fragment length polymorphism (T-RFLP). Their study found a notable increase in the number of _C23O_ gene copies during the biodegradation of 4-n-NP, representing the first genetic evidence of a potential aromatic ring meta-cleaving pathway for NP. On the other hand, the alkane hydroxylases (_alk_) gene and single-component monooxygenase (_sMO_) are also involved in the degradation of 4-n-NP. Both were studied in _Stenotrophomonas_ strain Y1 and _Sphingobium_ strain Y2 using quantitative PCR (qPCR) [56]. The _alkB_ gene acts via the oxidation of the alkyl chain, while the _sMO_ gene is responsible for converting NP to hydroquinone via the ipso substitution pathway [57]. The relative abundance of the _alkB_ gene increased in the 4-n-NP biodegradation test in the presence of _Stenotrophomonas_ strain Y1. However, when _Splingobium_ strain Y2 was inoculated, the proportion of the _sMO_ gene increased instead of the _alkB_ gene. Also, [PERSON] et al. [58] identified a nonylphenol monooxygenase gene (_nmoA_) responsible for ipso-hydroxylation in the NP biodegradation conducted by _Pseudomonas putida_. In another previous study, the _alk_, _C12O_, _C23O_, _sMO_, and multi-component phenol hydroxylase (\(\alpha\)-subunit) (_mPH_) gene were investigated through PCR in a 4-n-NP-degrading bacterial consortium primarily composed of members from the genera _Splingomonas_, _Pseudomonas_, _Alicyclophilus_, and _Acidovorax_.