Enhanced Metabolic Mechanism Of Citrobacter Freundii By Selective Co-doped Double-layerα-Fe₂O₃ Nanorod Arrays

Light and chemical energy serve as essential nutrients and energy sources for all living organisms on Earth.Non-photosynthetic microorganisms can directly or indirectly obtain energy from sunlight through inorganic media such as semiconductor minerals.Photoelectrons generated by semiconductors enter microbial cells and participate in the material cycle and energy metabolism of microorganisms either directly or indirectly.The extensive interaction between microorganisms and inorganic media has significantly impacted the development and evolution of terrestrial organisms.The investigation of the distinctive electron transfer mechanism employed by electrically active microorganisms,along with the establishment of metabolic features and energy utilization models of electrophilic microorganisms,holds paramount importance for understanding this particular type of metabolism.Such knowledge bears practical implications for exploring the unique reduction capacity of electrophilic microorganisms and their potential to utilize electrical energy directly.In this study,fluorine-doped SnO₂ conductive glass（SnO₂:F,FTO）was employed as a substrate to synthesize cobalt（Co）-doped dual-layerα-Fe₂O₃（hematite）nanorod arrays via a two-step hydrothermal method.The photoelectric characteristics of the synthesized hematite semiconductors were investigated.In addition,the electrochemical characteristics of hematite photo-generated electrons participating in the valence transition of Cr（VI）and the kinetic characteristics of photoelectron transfer were also explored.Using Citrobacter freundii as the model electrically active microorganism,a double-chamber bioelectrochemical system（BES）was established to examine the effects of semiconductor photoelectrons on the metabolism of electrophilic microorganisms and their intracellular transmission mechanisms using general,electrochemical,and histological methods.The main content and results of the study are as follows:（1）Single-layerα-Fe₂O₃ nanorod arrays,Co-dopedα-Fe₂O₃ nanorod arrays,and dual-layerα-Fe₂O₃ nanorod arrays selectively doped with Co in the upper and/or lower layers were synthesized separately using the hydrothermal method.The crystal morphology of Fe₂O₃nanorod arrays was characterized by scanning electron microscope（SEM）,X-ray diffraction（XRD）,X-ray photoelectron spectroscopy（XPS）,and UV-visible diffuse reflectance spectrum（UV-DRS）.Their photoelectric response ability was tested using electrochemical workstations,and the photocatalytic reduction of Cr（VI）was employed to further evaluate their catalytic performance.The results demonstrated that the synthesizedα-Fe₂O₃ has an obvious nanorod structure,and its length changes with Co doping.α-Fe₂O₃ exhibits p-type semiconductor characteristics and is consistent with typical hematite,with increased light absorption range,optical response-ability,and optical current density.The dual-layerα-Fe₂O₃ thin film effectively improves the photoelectric performance of the electrode,and bottom Co doping further enhances catalytic performance,reaching a current density of 1.37 m A/cm².It was preliminarily determined to have good catalytic performance for the photoelectric reduction of Cr（VI）.（2）Electrochemical methods were employed to determine the optimal conditions for the electrode reduction of Cr（VI）,to investigate the redox process of Cr（VI）,and to establish the optimal conditions and reaction kinetics of selective Co-dopedα-Fe₂O₃ photoelectrodes.The influences of typical hole trapping agents,p H,and dissolved oxygen conditions on the photocatalytic reduction of Cr（VI）byα-Fe₂O₃ nanorod arrays were examined.The results indicated that Cr（VI）primarily undergoes a two-step single-electron reduction process to convert to Cr（III）,while the oxidation process directly oxidizes Cr（III）to Cr（VI）.The p H value impacts the morphology of Cr（VI）in aqueous solutions and the photoelectric performance of the electrode.The investigation of reaction kinetics revealed that upon adding hole-trapping agents,electrolyte resistance（Rs）and electron transfer resistance（Rct）decreased,while electron transfer speed increased.With an FTO at E_bias=1.0 V vs.SCE and a light intensity of100 m W/cm²,the synergistic rate of photocatalytic removal of Cr（VI）withα-Fe₂O₃:Co/α-Fe₂O₃ double-layer composite electrode reached 179.4%.By incorporating citric acid and acetic acid as hole-trapping agents,the removal rate of Cr（VI）by photocatalysis for 3 h increased by 2.09 times and 1.3 times,respectively.A comprehensive analysis suggested that doping Co in the lower layer can reduce the recombination of internal and surface charges and holes inα-Fe₂O₃,improve carrier transfer,accelerate surface reaction kinetics,and result in changes in Fermi energy levels,significantly enhancing electrochemical performance.（3）An H-type dual chamber three-electrode bioelectrochemical system was established.The system used a double layer composite electrode with the best photoelectric response as the anode electrode material.In addition,C freundii was used as the research object of cathode electroactive microorganisms,and the photoelectrode pair effects were investigated on the growth and metabolism of C freundii.The results showed that photoelectrons had a significant growth-promoting effect during the logarithmic phase of the growth of electrically active microorganisms.This significantly shortened the time for the bacteria to reach the stable phase.3D fluorescence spectroscopy analysis revealed substantial differences in the metabolites of C.freundii compared to the control at 48 h,with notable disparities in certain organic acids and protein metabolism.High-performance liquid chromatography confirmed the differences in the metabolism of some organic acids in C.freundii,and metabolomic methods further investigated the variations in protein and organic acid metabolism in log-phase bacteria.（4）An H-type dual-chamber bioelectrochemical system was established to further examine the molecular mechanism of photoelectron-promoted electroactive microbial growth.The microbial films on cathodic graphite plates were electrochemically characterized to explore the changes in the electrochemical activity of C.freundii.Transcriptome analysis was conducted to investigate the transcriptional effects of photoelectrons on C.freundii.The results revealed that hematite photoelectrons altered the electrochemical activity of the C.freundii bacterial surface.Transcriptome results showed differential expression of NADH,acetyl coenzyme A,and others in C.freundii.The two-component system,pyruvate metabolic pathway,and oxidative phosphorylation are the primary transcriptional pathways by which photoelectrons influence differentially expressed genes in C.freundii.A preliminary analysis of the electron transfer mechanism of photoelectron uptake in C.freundii was performed by combining the above electrochemical tests with metabolomic and transcriptomic results.Through the above research,new ideas have been provided for the absorption and utilization of photoelectrons by electrophilic microorganisms,and a foundation has been laid for subsequent related applications.