Carbon : nitrogen : phosphorus ratios influence biofilm formation by Enterobacter cloacae and Citrobacter freundii

To test the effects of C : N : P ratio modification of a well-known nutrient medium formulation, the Endo formulation on biofilm formation by Enterobacter cloacae Ecl and Citrobacter freundii Cf1 in both single-species and binary species biofilms. The C : N : P atom : atom ratio of a well-known nutrient medium formulation, the Endo formulation, that has been applied in fermentative biohydrogen studies, was modified to include two different C concentrations, one containing 17·65 g l⁻¹ and the other 8·84 g l⁻¹ sucrose, each containing four different C : N : P ratios, two at higher C : N : P ratios (334 : 84 : 16·8 and 334 : 84 : 3) and two at lower C : N : P ratios (334 : 28 : 5·6 and 334 : 28 : 1). Attached cells were enumerated after dislodging the biofilms that had formed on granular activated carbon (GAC). The modified medium containing 17·65 g l⁻¹ sucrose and having a C : N : P ratio of 334 : 28 : 5·6 resulted in significantly (P < 0·05) higher counts of attached cells for both single-species biofilms at 7·73 log₁₀ CFU g⁻¹ GAC and 9·3 log₁₀CFU g⁻¹ GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively, and binary species biofilms at 8·2 log₁₀ CFU g⁻¹ GAC and 6·34 log₁₀ CFU g⁻¹ GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively. Scanning electron micrographs showed qualitative evidence that the 334 : 28 : 5·6 ratio encouraged more complex and extensive biofilm growth for both single-species and binary species biofilms. The differences in the attachment numbers between the different ratios were found not to be a result of the individual actions of the bacterial isolates involved but rather because of the effects of the various C : N : P ratios. The 334 : 28 : 5·6 ratio showed significantly (P < 0·05) higher counts of attached cells for both single-species and binary species biofilms. This study indicates that C : N : P ratios should be a key consideration with regard to maximizing biofilm formation in shake flask and fluidized bed bioreactor studies as well as understanding fundamental factors affecting biofilm growth in natural environments.