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This study aimed to increase bacterial growth and 5-aminolevulinic acid (ALA) biosynthesis of Rhodobacter sphaeroides in wastewater treatment through adding ferrous ion (Fe2+).Results demonstrated that Fe2+ effectively enhanced the biomass production and ALA yield of R.sphaeroides.Moreover,the optimal Fe2+ dosage was found to be 400 μmol/L,which was associated with the highest biomass of 4015.3 mg/L and maximum ALA yield of 15.9 mg/g-dry cell weight (mg/g-DCW).Mechanism analysis revealed that Fe2+ vastly improved Adenosine Triphosphate (ATP) production by up-regulating the nif gene expression,and increasing ATP enhanced the biomass and ALA yield by supplying energy for bacterial growth and ALA biosynthesis,respectively.Correlation analysis showed that the ALA and ATP yields had positive relation with nifA and nifU gene expression.In addition,the nifA and nifU gene expression displayed high consistency of co-transcription at the optimal Fe2+ dosage.