Planktonic-cell yield of a pseudomonad biofilm

Abstract

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10(9) cells ml(-1), suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.

FIG. 1.

(A) Growth rate of Pseudomonas sp. strain CT07 in batch culture (▴) compared to the rate of attachment and growth typically observed on a glass surface (▪) during the early stages of biofilm formation. After inoculation and a 30-min period without flow, flow was resumed for 12 h, after which the accumulation at the glass surface was quantified at 30-min intervals (time zero and onward on the x axis). (B) Numbers of culturable biofilm-detached cells in the effluent of flow cells with total attachment areas of 500 (□) and 1,400 (▪) mm², respectively. (C) Effect of carbon source and nutrient type on the number of culturable biofilm-detached cells in the effluent associated with 3-day-old biofilms. MSS, minimal salt solution.

FIG. 2.

Attachment and biofilm accumulation by Pseudomonas sp. strain CT07 at a Plexiglas surface over a period of 70 h. This plot shows the average of the two replicate experiments.

FIG. 3.

(A) Viability of control (□) and biocide-treated (▪) cells as determined with BacLight viability staining. There was a significant difference between the sensitivity (percent reduction in viable numbers) of sessile and biofilm-detached cells and sessile and planktonically grown (chemostat) populations. (B) Culturability of control (□) and biocide-treated (▪) cells. The cells derived from biofilms were more sensitive than chemostat-derived cells; the effect on viability was similar to that shown in panel A (D; dilution rate). (C) Percent area coverage by biofilms and released biofilm biomass before (□) and after (▪) biocide treatment. To determine the extent of detached biofilm biomass, flow cell effluent was stained with BacLight and a standardized volume was immobilized on filters, followed by measuring the area coverage using image analysis. The difference in percent reduction in area coverage between the attached and flow cell effluent cells was significant (P = 0.026).