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.2022 Apr 12;10(4):801.
doi: 10.3390/microorganisms10040801.

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

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

Oscar Kruppa et al. Microorganisms..

Abstract

Planctomycetes such asPlanctopirus 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 ofP. 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 cultivateP. 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.

Keywords: C:N ratio; MTT assay; Planctomycetes; Planctopirus limnophila; biofilm formation; chemically defined medium; crystal violet staining; fixed-bed cultivation; metal ions; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effects of different C:N ratios onP. limnophila growth and biofilm formation in microtiter plates. (A) MTT assay results (absorbance readings at 550 nm). (B) Crystal violet absorbance readings at 590 nm. (C) OD600 readings in the liquid phase. (D) Distribution of the CDW (g/L) between the carriers and liquid phase. Data are means ± standard deviations (n = 3 biological replicates). Statistical analysis for MTT assay, OD600 and CV staining was based on a two-samplet-test compared to control values (D1ASO), *P < 0.05.
Figure 2
Figure 2
Effects of different Fe, Cu, and Zn concentrations (Table 2) onP. limnophila growth and biofilm formation in microtiter plates. (A) MTT assay results (absorbance readings at 550 nm). (B) Crystal violet absorbance readings at 590 nm. (C) OD600 readings in the liquid phase. (D) Distribution of the CDW (g/L) between the carriers and liquid phase. Data are means ± standard deviations (n = 3 biological replicates). Statistical analysis for MTT assay, OD600 and CV staining was based on a two-samplet-test compared to control values (D1ASO medium), *P < 0.05.
Figure 3
Figure 3
Effects of different H2O2 concentrations (5 µM to 50 mM) onP. limnophila growth and biofilm formation in microtiter plates. (A) MTT assay results (absorbance readings at 550 nm). (B) Crystal violet absorbance readings at 590 nm. (C) OD600 readings in the liquid phase. (D) Distribution of the CDW (g/L) between the carriers and liquid phase. Data are means ± standard deviations (n = 3 biological replicates). Statistical analysis for MTT assay, OD600 and CV staining was based on a two-samplet-test compared to control values (D1ASO medium), *P < 0.05.
Figure 4
Figure 4
Effects 50 µM Fe or Cu, or a low C:N ratio (5.7:1) onP. limnophila growth and biofilm formation in a fixed-bed bioreactor. (A) OD600 in the liquid phase (conditioning vessel). (B) MTT assay results (absorbance readings at 550 nm). (C) Distribution of the CDW (g/L) between the carriers and liquid phase. Data in (B,C) are means ± standard deviations (n = 3 biological replicates). Statistical analysis for MTT assay was based on a two-samplet-test compared to control values (D1ASO medium).
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References

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