Paracoccus sp. KDSPL–02 can use penicillin G as the sole carbon and energy source
First, the growth of Paracoccus when using penicillin as the sole carbon and energy source was investigated. As shown in Figure 1, when using glucose, sucrose or starch as the single carbon source, the growth of Paracoccus was better, and the OD600 after 6 h of growth reached 1.2, 0.9 and 0.9, respectively. When penicillin was used as the sole carbon and energy source, the growth of Paracoccus was relatively poor with the OD600 reaching 0.4 after 6 h of growth. Although the growth status of Paracoccus was poor when using penicillin, this showed that Paracoccus could still survive.
Most microorganisms are not known to use antibiotics as sole carbon and energy sources; co-metabolism is often required to maintain growth. In our study, Paracoccus sp. KDSPL–02 could advantageously use penicillin as the sole carbon and energy source. This ability is necessary for degrading antibiotics. The biomineralization of an antibiotic is the complete metabolism of the antibiotic, i.e., bacterial cells first decompose the antibiotic into small molecular intermediate degradation products that can be used by the bacteria through specific degradative enzymes. These intermediate degradation products then pass the central bacterial metabolic pathway for breakdown into carbon dioxide and water to realize the complete degradation of antibiotics.
Optimization of penicillin biotransformation by paracoccus sp. KDSPL–02
Because penicillin hydrolyses in water, penicillin biotransformation in solution cannot be directly measured. Hence, all experiments included control treatment reactors that contained no cells. Only hydrolysis occurred in the control reactors, whereas both hydrolysis and biotransformation occurred in the bacterial treatment reactors. Observed differences in penicillin concentrations with the control reactors were then attributed to biotransformation.
1) Effects of paracoccus sp. KDSPL–02 cell dry weight on penicillin biotransformation
The biodegradation conditions were optimized by single-factor tests under different conditions, such as paracoccus sp. KDSPL–02 cell dry weight (5 g/L- 20 g/L). The research result showed that the influence of the paracoccus sp. KDSPL–02 cell dry weight on the degradation was remarkable. The degradation rate increased steadily with increasing cell dry weight. When the cell activity was above 10 g/L, the degradation rate reached to 100% within 12 h (Figure 2a).
2) Effects of the concentration of the substrate on penicillin biotransformation
The degradation also depended strongly on the concentration of the substrate. As the initial concentration of penicillin G increased (from 0.4 g L–1 to 5.2 g L–1), the degradation ability of the strain lost gradually (Figure 2b). The degradation rate of penicillin G was approximately 100% in the beginning, and then decreased as the concentration of penicillin G increased, becoming inhibited above 2.0 g L–1. The initial highest concentration of penicillin G optimized was 0.8–1.6 g L–1.
Earlier reports on penicillin G biodegradation by different bacterial strains are summarized in Table S1. The 100% degradation rate presented in this study was comparable to or higher than those in previous studies. For instance, Phanerochate chrysosporium tolerates high penicillin G concentrations up to 2000 mg L–1 but can only achieve a degradation efficiency of 61.4% (Hathroubi et al., 2015). Furthermore, the penicillin G degradation efficiencies of Klebsiella pneumoniae Z–1, Actinobacillus pleuropneumoniae, and Achromobacter sp. CCM 2428 reach 99.9% or nearly 100% at penicillin G concentrations below 300 and 500 mg L–1, respectively, but an increase in tolerance limits the degradation ability (Skrob et al., 2003; Kumar et al., 2012; Wang et al., 2015). Compared with the above bacterial strains, Paracoccus sp. KDSPL–02 has significant advantages. The free cells completely eliminated penicillin G when its initial concentration was 1.2 g L–1; the cells retained their degradation efficiency to 2.0 g L–1, and the final degradation product was benzoic acid.
3) Effects of temperature and pH on penicillin biotransformation
Biodegradation conditions were optimized by single-factor tests at a temperature range from 25°C to 40°C and a pH range from 4.0 to 10.0. To eliminate interference from penicillin hydrolysis on the detection of penicillin biodegradation, we investigated the hydrolysis of penicillin under different conditions.
The temperature and pH of the degradation reaction are two important factors. The degradation rate steadily increased as the temperature increased from 25°C to 30°C. When the temperature was raised to 40°C, degradation efficiency declined. At 1.2 g L–1, penicillin G was almost completely degraded by whole-cell catalysis from 30°C–35°C (Figure 2c).
Because pH changes with degradation, the initial pH of the degradation solution containing penicillin strongly impacted the reaction rate. The effect of the initial pH is plotted in Figure 2d. The data shows that the degradation rate was lowest at pH 5.0, which indicated that the KDSPL–02 cell enzymes did not function well under acidic conditions. The degradation activity was higher at pH 9.0, indicating that KDSPL–02 whole-cell catalytic degradation of penicillin G occurred under neutral or weakly alkaline conditions, as confirmed in previous studies (Anwar et al., 2009). The highest degradation rate was achieved from pH 7.0–9.0, which indicated that Paracoccus sp. KDSPL–02 was functional over a wide pH range. Additionally, we found that penicillin was unstable in aqueous alkaline solution (when the pH is greater than 8.0) and obvious hydrolysis will occur. Hydrolysis was not observed in acidic (pH 4.0–7.0), neutral (pH 7.0) and weakly alkaline (pH 7.0–8.0) environments.
Pathway analysis of penicillin G biodegradation by parococcus sp. KDSPL–02
The process of penicillin G degradation via biocatalysis is generally very complex, a few intermediates were shown by HPLC when penicillin G was treated with whole cell KDSPL–02 (Figure S1). Penicillin G in the medium solution without whole cell KDSPL–02 have not shown obvious degradation detected by HPLC. In addition, the number of the intermediates and the values varied with penicillin G concentration. Three peaks appeared in the HPLC when the concentration was below 0.8 g L–1, and the change of each component with the reaction carrying out shown in Figure 3a. While the concentration of penicillin G was at 1.6 g L–1, five components exhibited in the HPLC, and the conversions were shown in Figure 3b.
All intermediates attributed to peaks in HPLC were isolated and collected to yield purified samples. The structures of the compounds were determined via 1H NMR, 13C NMR, and LC–MS. The intermediate compounds corresponding to peak 2 and 3 had the same mass spectrum, the molecular ion peak [M+1]+ (m/z) was 353.2, and no fragment signal peaks showed a difference in LC–MS (Figure S2a). Moreover, their 1H NMR and 13C NMR spectra were highly similar, belonging to potassium 2-(carboxy(2-phenylacetamido)methyl)–5,5-dimethylthiazolidine–4-carboxylate (penicilloic acid) and potassium 2-(4-carboxy–5,5-dimethylthiazolidin–2-yl)–2-(2-phenylacetamido) acetate (potassium penicilloic acid) (Figure S3a-b). The former is the product formed by opening the ring of β-lactam in penicillin G, while the latter is its isomer that was formed through the exchange of an H+ with potassium ion. The compound corresponding to peak 4 is simple. The LC–MS showed that the molecular ion peak [M+1]+ (m/z) was 137.3 (Figure S2b), and 1H NMR and 13C NMR indicated the presence of a benzene ring in its structure. Thus, this peak was belonged to phenylacetic acid (Figure S4a-b). For the species corresponding to peak No. 5, LC–MS showed a molecular ion peak [M+1]+ of 235.1 (Figure S2c). Moreover, the characteristic structure of 5,5-dimethylthiazolidine contained in penicillin G was still present in its 1H NMR and 13C NMR spectra (Figure S5a-b). Thus, this peak in HPLC belonged to the potassium salt of 2-(amino(carboxyl)methyl)–5,5-dimethylthiazolidine–4-carboxylic acid and (compound 5) potassium 2-amino–2-(4-carboxy–5,5-dimethylthiazolindin–2-yl) acetate (compound 6)..
According to the HPLC，MS and HMR analysis of the intermediates, the possible pathway of complete degradation process catalysed by KDSPL–02 whole cells was proposed, as shown in Figure 4. β-Lactam hydrolase initially catalyzed penicillin G to open the β-lactam ring, forming intermediate penicilloic acid, which was simultaneously and reversibly converted with intermediate potassium penicilloic acid. Afterward, the open ring compounds were hydrolyzed by amide hydrolase to form phenylacetic acid and compound 5 and compound 6. Phenylacetic acid was oxidized by oxidases in the cell through oxophenylacetic acid 7 which coincide with the peak 150.1 in the MS of No.4 degradation mixture to form inorganic mineralizers. Compound 5, which coincide with the peak 271.1 in the MS of its degradation mixture, was initially converted to the corresponding α-keto acid 8, which then underwent further decarboxylation, oxidation, and hydrolysis to yield small inorganic compounds.
Light enhancing the penicillin biodegradation efficiency of Paracoccus sp. KDSPL–02.
Paracoccus sp. KDSPL–02 was a more effective catalyst of penicillin G degradation under the optimized conditions. Using the same methods for the treatment of penicillin G in solution, wastewater contained penicillin G was treated. The results showed that 0.8–1.6 g L–1 penicillin G was almost degraded completely by whole-cell catalysis at 30°C. However, in the degradation of penicillin G, in solution or wastewater, the overall reaction rate was subject to secondary reactions. To improve the biodegradation efficiency, visible light irradiation was used to enhance the biocatalytic degradation.
Changes in species concentrations with treatment time during the degradation of penicillin G with an initial concentration of 1.2 g L–1 are shown by HPLC data in Figure 5a. Penicillin G concentration decreased rapidly with degradation reaction. Peak No. 1 (penicillin G) completely disappeared after 5 h at an initial concentration below 1.2 g L–1. However, the conversion rate of penicilloic acid intermediate was slow and required 24 h. Phenylacetic acid and compound 5 intermediates degraded more rapidly than penicilloic acid within 18 h. In the degradation process, the rate-limiting reaction was the conversion of penicilloic acid into phenylacetic acid and compound 5. To improve the degradation kinetics, we tested auxiliary strategies. The results indicated that visible light markedly accelerated the rate-limiting reaction. Based on HPLC data (Figure 5b), the degradation time decreased from 24 h to 18 h. The effect of photocatalysis on penicillin G degradation was further studied. The data shown in Table 1 indicates that synergistic catalysis can accelerate the rate of the treatment, i.e., the conversion of penicilloic acid or the conversion of phenylacetic acid and compound 5 into small inorganic molecules. Regarding the heating effect of visible light, the same results were obtained when homogeneous LED lights, such as green and blue, were substituted for visible light. Therefore, the heating effect was not a major factor in the degradation process.
Phenylacetic acid removal during visible light treatment
Phenylacetic acid intermediate often accumulates, especially when treating high concentrations of penicillin G, and exhibits a strong inhibitory effect that could inhibit the growth of the strain as shown in Figure 6a. Although the function of visible light in co-catalysis was not clearly elucidated, the mechanism by which it induces oxidation was determined. Thus, we speculated that visible light enhances the oxidation of phenylacetic acid to reduce biodegradation inhibition. Experimental results (Figure 6b) indicated that the rate of phenylacetic acid oxidation can be accelerated by visible light.
Photocatalysts are important components in traditional photobiological degradation processes. Several commonly photocatalysts used are listed in Table S2. In our study, the penicillin treatment process was accomplished using light-irradiation assisted biocatalysts (Paracoccus sp. KDSPL–02) without other photocatalysts. Penicillin was biodegraded by enzymes from Paracoccus sp. KDSPL–02. Light irradiation played an important role to accelerate the biodegradation rate of Paracoccus sp. KDSPL–02. We speculated that several enzyme candidates in the oxidation process could respond to light irradiation. The traditional photocatalysis process requires a photo-oxidant. In the process of photooxidation, the substrate usually transforms to a hyper-oxidation product . These hyper-oxidation products cannot be further eliminated, which may cause secondary pollution. Due to the unique light sensitivity of Paracoccus sp. KDSPL–02, penicillin biodegradation activated by light was achieved. Because no additional photocatalysts were added in the combined biodegradation and photocatalysis process, the process for separation was simplified, which reduced production costs. This process is more efficient and environmentally friendly.
Further research could greatly expand the scope of application of the bacterial strain, especially in the treatment of antibiotic waste waters, industrial bacterial residue, and rivers around pharmaceutical factories. In terms of the biodegradative inhibition of benzoic acid in KDSPL–02, in situ product removal has shown advantages in improving production [39, 40], Further research is warranted to determine whether these advantages are applicable to biodegradation.
The whole genome of paracoccus sp. KDSPL–02 was analysed. The PPA pathway from bacteria can degrade phenylacetic acid, and the degradation product can be introduced to the central metabolism of microorganisms for mineralization . In genomic mining, several enzymes involved in the PPA pathway were discovered as shown in Table S3. Enzymes PaaN and PaaD were both found in the genome of Paracoccus sp. KDSPL–02. We speculated that light irradiation could promote the transcription and expression of these enzymes to further accelerate the biodegradation rate of phenylacetic acid.