Presentation Information
[P02-241]Development of a polyphosphate-hyperaccumulating Pseudomonas strain for phosphate removal from wastewater
○Tomohiro Morohoshi1, Yuri Hasegawa1, Masahiro Iwamoto1, Wataru Takahashi1, Nobutaka Someya2 (1. Utsunomiya University (Japan), 2. National Agriculture and Food Research Organization (Japan))
Keywords:
polyphosphate,Pseudomonas,wastewater,water treatment,inorganic phosphate
[Purpose]
Inorganic phosphate (Pi) is a significant contributor to eutrophication, and enhancing bacterial polyphosphate (polyP) accumulation is a promising strategy for removing Pi from wastewater. Previously, we demonstrated that elevated polyP accumulation can be achieved by increasing the dosage of genes in the Pi-specific transport system (Pst) in Escherichia coli. Furthermore, we reported that a mutation in phoU, which encodes a negative regulator of Pst expression, resulted in high levels of polyP accumulation in E. coli. However, E. coli exhibits limited growth and metabolic activity in wastewater, rendering it unsuitable for wastewater treatments. Pseudomonas species exhibit numerous beneficial capabilities required for environmental biotechnology, including plant growth promotion, bioremediation, and wastewater treatment. This study aimed to identify polyP-hyperaccumulating strains among Pseudomonas putida and Pseudomonas fluorescens and to evaluate whether introducing a phoU mutation could further enhance Pi removal efficiency.
[Method]
A total of 79 strains of P. putida and P. fluorescens were screened for intracellular polyP levels. The type strain P. putida JCM 13063, with a completely sequenced genome, was selected for genetic modification. A non-marker phoU deletion mutant was constructed via two-step homologous recombination. PolyP accumulation was compared between the wild type and phoU mutant. Both strains were then cultured in artificial wastewater, and the Pi concentration in the medium was measured every 2 h to assess the removal kinetics.
[Results]
Most Pseudomonas strains accumulated higher levels of polyP than did E. coli. The phoU mutant of JCM 13063 accumulated approximately 20-fold more polyP than the wild type. In artificial wastewater, the phoU mutant removed all 0.2 mM Pi within 4 h, whereas the wild type did not achieve complete removal in the same timeframe.
[Consideration]
Pseudomonas species possess metabolic characteristics that are conducive to wastewater treatment. Their capacity to accumulate polyP indicates their significant potential for Pi removal. The deletion of phoU in JCM 13063 enhanced polyP accumulation, paralleling the effects previously documented in E. coli, albeit in a more environmentally resilient host. These findings suggest that integrating inherent metabolic advantages with targeted genetic modifications can markedly enhance Pi removal efficiency.
[Conclusion]
P. putida and P. fluorescens both showed a markedly higher capacity for polyP accumulation than E. coli. In particular, disruption of the phoU gene in P. putida JCM 13063 led to an even greater increase in polyP accumulation and enabled the efficient removal of Pi from synthetic wastewater. This gene deletion strategy holds promise for enhancing Pi removal efficiency in Pseudomonas, highlighting its potential utility in wastewater treatment applications.
Inorganic phosphate (Pi) is a significant contributor to eutrophication, and enhancing bacterial polyphosphate (polyP) accumulation is a promising strategy for removing Pi from wastewater. Previously, we demonstrated that elevated polyP accumulation can be achieved by increasing the dosage of genes in the Pi-specific transport system (Pst) in Escherichia coli. Furthermore, we reported that a mutation in phoU, which encodes a negative regulator of Pst expression, resulted in high levels of polyP accumulation in E. coli. However, E. coli exhibits limited growth and metabolic activity in wastewater, rendering it unsuitable for wastewater treatments. Pseudomonas species exhibit numerous beneficial capabilities required for environmental biotechnology, including plant growth promotion, bioremediation, and wastewater treatment. This study aimed to identify polyP-hyperaccumulating strains among Pseudomonas putida and Pseudomonas fluorescens and to evaluate whether introducing a phoU mutation could further enhance Pi removal efficiency.
[Method]
A total of 79 strains of P. putida and P. fluorescens were screened for intracellular polyP levels. The type strain P. putida JCM 13063, with a completely sequenced genome, was selected for genetic modification. A non-marker phoU deletion mutant was constructed via two-step homologous recombination. PolyP accumulation was compared between the wild type and phoU mutant. Both strains were then cultured in artificial wastewater, and the Pi concentration in the medium was measured every 2 h to assess the removal kinetics.
[Results]
Most Pseudomonas strains accumulated higher levels of polyP than did E. coli. The phoU mutant of JCM 13063 accumulated approximately 20-fold more polyP than the wild type. In artificial wastewater, the phoU mutant removed all 0.2 mM Pi within 4 h, whereas the wild type did not achieve complete removal in the same timeframe.
[Consideration]
Pseudomonas species possess metabolic characteristics that are conducive to wastewater treatment. Their capacity to accumulate polyP indicates their significant potential for Pi removal. The deletion of phoU in JCM 13063 enhanced polyP accumulation, paralleling the effects previously documented in E. coli, albeit in a more environmentally resilient host. These findings suggest that integrating inherent metabolic advantages with targeted genetic modifications can markedly enhance Pi removal efficiency.
[Conclusion]
P. putida and P. fluorescens both showed a markedly higher capacity for polyP accumulation than E. coli. In particular, disruption of the phoU gene in P. putida JCM 13063 led to an even greater increase in polyP accumulation and enabled the efficient removal of Pi from synthetic wastewater. This gene deletion strategy holds promise for enhancing Pi removal efficiency in Pseudomonas, highlighting its potential utility in wastewater treatment applications.
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