Presentation Information
[P04-511]Visualization of Metabolic Activity in Mixed-Species Biofilms of Shewanella oneidensis and Paracoccus denitrificans
○Mei Koshimizu1, Hiromasa Tongu1, Masanori Toyofuku2,3,4, Nobuhiko Nomura2,3,4,5, Yoshihide Tokunou2,6 (1. Grad. Sch. Life Environ. Sci., Univ. Tsukuba (Japan), 2. Fac. Life Environ. Sci., Univ. Tsukuba (Japan), 3. MiCS, Univ. Tsukuba (Japan), 4. TIAR, Univ. Tsukuba (Japan), 5. TARA, Univ. Tsukuba (Japan), 6. NIMS (Japan))
Keywords:
Extracellular Electron Transfer,3D imaging of biofilm,redox,interspecies interaction,wastewater treatment
[Purpose]
In microbial industries, including wastewater treatment, mixed microbial systems are widely employed. To improve their efficiency, interactions within biofilms, such as cross-feeding and quorum sensing, have been extensively studied. In contrast to these molecule-mediated interactions, we have demonstrated a novel metabolic regulatory mechanism based on electron flow. In biofilms, oxygen concentration decreases at the bottom distant from the surface, resulting in lower metabolic activity of aerobic bacteria. We previously revealed that Shewanella oneidensis, a bacterium that respires oxygen, transfers electrons from the bottom to the surface of biofilms across distances exceeding its cell length, enabling the oxidation of NADH to NAD+ at the bottom layer of cells. It is suggested that electron transfer within biofilms is linked to bacterial survival strategies. However, such electron transfer has been observed in S. oneidensis monoculture biofilms, it is not clear how electron transfer occurs in mixed-species biofilms. In this study, we analyzed electron transfer within mixed-species biofilms formed with Paracoccus denitrificans, a bacterium that has been actively studied in co-culture systems in the field of wastewater treatment.
[Method]
A plasmid encoding a fluorescent reporter for the intracellular NADH/NAD+ ratio was introduced into S. oneidensis. Mixed-species biofilms with P. denitrificans were formed on agar media, and the spatial distribution of the NADH/NAD+ ratio in S. oneidensis was analyzed by observing fluorescence using two-photon excitation microscopy. Electron transfer within the mixed-species biofilms was evaluated using electrochemical measurements.
[Results]
In mixed-species biofilms, the intracellular NADH/NAD+ ratio of S. oneidensis in deeper layers was significantly lower than in monocultures, indicating maintained metabolic activity. In contrast, a mutant lacking outer membrane cytochrome c, which is involved in electron transfer within biofilms, showed a significantly higher NADH/NAD+ ratio than the wild type. Additionally, electron transfer activity of S. oneidensis cell was higher in the mixed-species system.
[Consideration]
These results suggest that electron transfer via outer membrane cytochrome c of S. oneidensis functions in mixed-species biofilms and contributes to maintaining metabolic activity in deeper layers. Notably, both electron transfer and metabolic activity were enhanced in the mixed-species system, possibly due to interactions with P. denitrificans, such as metabolite exchange.
[Conclusion]
This study demonstrated that electron transfer within biofilms by S. oneidensis functions in mixed-species systems and that electron flow may act as a mechanism for spatial regulation of metabolic activity within biofilms. These findings may be applicable not only to wastewater treatment but also to the development of novel technologies for controlling microbial communities at the collective level.
In microbial industries, including wastewater treatment, mixed microbial systems are widely employed. To improve their efficiency, interactions within biofilms, such as cross-feeding and quorum sensing, have been extensively studied. In contrast to these molecule-mediated interactions, we have demonstrated a novel metabolic regulatory mechanism based on electron flow. In biofilms, oxygen concentration decreases at the bottom distant from the surface, resulting in lower metabolic activity of aerobic bacteria. We previously revealed that Shewanella oneidensis, a bacterium that respires oxygen, transfers electrons from the bottom to the surface of biofilms across distances exceeding its cell length, enabling the oxidation of NADH to NAD+ at the bottom layer of cells. It is suggested that electron transfer within biofilms is linked to bacterial survival strategies. However, such electron transfer has been observed in S. oneidensis monoculture biofilms, it is not clear how electron transfer occurs in mixed-species biofilms. In this study, we analyzed electron transfer within mixed-species biofilms formed with Paracoccus denitrificans, a bacterium that has been actively studied in co-culture systems in the field of wastewater treatment.
[Method]
A plasmid encoding a fluorescent reporter for the intracellular NADH/NAD+ ratio was introduced into S. oneidensis. Mixed-species biofilms with P. denitrificans were formed on agar media, and the spatial distribution of the NADH/NAD+ ratio in S. oneidensis was analyzed by observing fluorescence using two-photon excitation microscopy. Electron transfer within the mixed-species biofilms was evaluated using electrochemical measurements.
[Results]
In mixed-species biofilms, the intracellular NADH/NAD+ ratio of S. oneidensis in deeper layers was significantly lower than in monocultures, indicating maintained metabolic activity. In contrast, a mutant lacking outer membrane cytochrome c, which is involved in electron transfer within biofilms, showed a significantly higher NADH/NAD+ ratio than the wild type. Additionally, electron transfer activity of S. oneidensis cell was higher in the mixed-species system.
[Consideration]
These results suggest that electron transfer via outer membrane cytochrome c of S. oneidensis functions in mixed-species biofilms and contributes to maintaining metabolic activity in deeper layers. Notably, both electron transfer and metabolic activity were enhanced in the mixed-species system, possibly due to interactions with P. denitrificans, such as metabolite exchange.
[Conclusion]
This study demonstrated that electron transfer within biofilms by S. oneidensis functions in mixed-species systems and that electron flow may act as a mechanism for spatial regulation of metabolic activity within biofilms. These findings may be applicable not only to wastewater treatment but also to the development of novel technologies for controlling microbial communities at the collective level.
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