# Biodegradation of Picolinic Acid by _Rhodococcus_ sp. PA18
## 3 Results
### Identification of PA Metabolites  
PA degradation by PA18 was analyzed preliminarily using ultraviolet-visible spectroscopy (UV-VIS). In addition to the parent compound, PA (maximum at 260 nm), one major metabolite was detected with an increase in the absorption at 310 nm (Figure 4A). The intermediate product was  
Figure 3: Effects of inocula (**A**) and the initial PA concentration (**B**) on the degradation of PA in MSM. The error bars show standard deviations.  
Figure 2: Utilization of PA during the growth of _Rhodococcus_ sp. PA18 in mineral salts medium (MSM) (pH 7.0). The strain was pre-cultured with PA (**A**). The strain was pre-cultured in a Luria Bertani (LB) medium (**B**).  
collected and subjected to high performance liquid chromatography (HPLC). The peak corresponding to PA was detected at a retention time of 4.1 min, and an unknown peak appeared at 8.3 min (Figure 4B).  
Then, the sample was further analyzed using liquid chromatography/time of flight-mass spectrometry (LC/TOF-MS). The fragment at \(m/z\) 124.0398 [M\({}^{+}\)H]\({}^{+}\) conformed to the molecular weight of 124.0354 for PA (Figure 5). In addition, another molecular ion of \(m/z\) 140.0346 [M\({}^{+}\)H]\({}^{+}\) was detected. An increment of 16 in the molecular weight indicated that an oxygen atom was added to the PA, which was consistent with the molecular weight of the predicted metabolite 6 HPA (C\({}_{6}\)H\({}_{5}\)NO\({}_{3}\), m/z 140.0303) (Figure 5). Therefore, the PA was possibly hydroxylated by PA18, which was consistent with the results of the previous reports on PA degradation by _Burkholderia_ sp. ZD1 [3] and _Alcaligenes faecalis_ JQ135 [12]. A standard sample of 6 HPA was also analyzed using HPLC under the same conditions, and its retention time was identical to that of the unknown peak mentioned above. Therefore, the intermediate metabolite was identified as 6 HPA. Throughout the degradation process, no other intermediates were detected.  
Figure 4: PA degradation analysis by the strain PA18 using UV-VIS and high-performance liquid chromatography (HPLC). (**A**) The UV-VIS absorption of each compound during PA degradation from 200 to 400 nm. (**B**) HPLC analysis of the conversion of PA into 6 HPA at 12 h. The black line indicates the sample collected at 0 h and the red line indicates at 12 h.  
Figure 5: LC/TOF-MS profiles of PA and 6 HPA.  
Usually, the first step in the microbial degradation of the aromatic ring or heterocyclic aromatic compounds is the introduction of a hydroxyl group, and these dehydroxylated substrates can be subsequently decarboxylated [27]. Finally, it can be easily cleaved by dioxygenase or mooxygenase via either an ortho-cleavage pathway or a meta-cleavage pathway, leading to the formation of tricarboxylic acid cycle intermediates [28; 29]. The first step, hydroxylation of pyridine or its derivatives, is the key step in biodegradation. Several studies have already shown that pyridine [30], PA [12; 31], nicotine [32], and quinolone [33] are hydroxylated at the carbon adjacent to the heteroatom. Pyridine derivatives possessing hydroxyl groups can be used as important starting materials for the synthesis of agrochemicals and pharmaceuticals [34; 35]. Future research will be focused on cloning the gene encoding hydroxylase, as well as further study of the enzymatic characteristics.