Results

Our preliminary design and engineering efforts have yielded promising experimental outcomes. The success of our engineering was substantiated by the analysis of the collected precipitate and the subsequent assay of its enzyme-like activity. Ultimately, we deployed our Ccm-promoter bioreactor for the degradation of lignin and corn stalks, achieving a notably enhanced degradation rate.

Our findings indicate that the culture medium supplemented with Mn²⁺ exhibits a distinct color variation. In a control experiment devoid of bacteria, there is no significant color change observed. However, in the presence of bacteria, the recombinant strain of P. putida displays a noticeably deeper brownish-yellow hue compared to the wild type. This coloration is likely attributable to the formation of high-valence manganese oxide (MnOx). Subsequently, the precipitate formed during the cultivation of P. putida was harvested.

The structural characteristics of the precipitate were meticulously examined using transmission electron microscopy (TEM) and X-ray diffraction (XRD), and their manganese catalase (MCO)-like activities were evaluated. The TEM images revealed that all samples are nanomaterials with distinct morphologies. Analysis of the XRD spectra indicated that Mn²⁺ was auto-oxidized to MnO in ambient conditions. The wild type P. putida facilitated the formation of Mn₂O₃, whereas the recombinant strain produced Mn₃O₄. Based on the activity assays, MnO exhibited negligible enzymatic activity. In contrast, Mn₂O₃ demonstrated typical enzyme-like activity, albeit significantly inferior to that of Mn₃O₄.

Figure 1. The images of Mn²⁺ oxidation without bacteria, with P. putida and ccm-promoter engineered P. putida.

Figure 2. The image of MnOx collected from different culture medium in Figure 1.

Figure 3. The TEM images of produced MnOx in Figure 2 and their XRD spectra.

Figure 4. The XRD spectra of produced MnOx in Figure2.

Figure 5. The laccase oxidase-like activity of produced MnOX and corresponding UV-vis absorbance spectra of reaction system.

Figure 6. The laccase oxidase-like activity of ccm-promoter engineered P. putida.

To evaluate our design, the lignin degradation performance of the recombinant P. putida bioreactor was tested by culturing it in a dealkaline medium. The concentration, morphology, structural features, and chemical composition were analyzed. Furthermore, P. putida was cultured with both raw and treated corn stalks to assess the potential generation of carbon sources following degradation. Collectively, these analyses demonstrated that the ccm-promoter engineered P. putida exhibits significantly enhanced degradation capabilities towards lignocellulose, thereby validating our successful engineering efforts. Moreover, the engineered P. putida could serve as an excellent platform for the utilization and transformation of lignocellulose, offering a promising pathway for the development of renewable energy sources.

Figure 7. The image of lignin-containing solution before and after treatment.

Figure 8. The time-dependent UV-vis spectra of dealkaline lignin-contained system.

Figure 9. The images of corn stalk before and after degradation.

Figure 10. SEM images of corn stalk before and after degradation

Figure 11. XRD and FTIR spectra of lignin before and after degradation.

Figure 12. The three-dimensional excitation-emission matrix fluorescence spectroscopy of the corn stalk along with reaction time.

Figure 13. The plate count results of P. putida cultured with raw lignin (a), M9 full medium (b) and treated corn stalk (c), and its corresponding colony count.