Screening for Biofilm-Stimulating Factors in the Freshwater Planctomycete Planctopirus limnophila to Improve Sessile Growth in a Chemically Defined Medium

Abstract

Planctomycetes such as Planctopirus limnophila offer a promising source of bioactive molecules, particularly when they switch from planktonic to sessile growth, but little is known about the corresponding biosynthetic gene clusters and how they are activated. We therefore screened for factors that promote sessile growth and biofilm formation to enable the cultivation of P. limnophila in a fixed-bed reactor. We carried out screening in microtiter plates focusing on biofilm formation and changes in optical density in response to various C:N ratios, metal ions, and oxidative stress. We used MTT assays and crystal violet staining to quantify biofilm formation. Positive factors were then validated in a fixed-bed bioreactor. The initial screen showed that D1ASO medium supplemented with NH4Cl to achieve a C:N ratio of 5.7:1, as well as 50 µM FeSO4 or CuSO4, increased the biofilm formation relative to the control medium. Exposure to H2O2 did not affect cell viability but stimulated biofilm formation. However, the same results were not replicated in the fixed-bed bioreactor, probably reflecting conditions that are unique to this environment such as the controlled pH and more vigorous aeration. Although we were able to cultivate P. limnophila in a fixed-bed bioreactor using a chemically defined medium, the factors that stimulate biofilm formation and inhibit planktonic growth were only identified in microtiter plates and further evaluation is required to establish optimal growth conditions in the bioreactor system.