Overview of the expression of transcriptional regulators known to affect biofilm in other <i>Clostridium</i> species.

<p>^a Expression 1 reflects the gene expression of the planktonic cells (no NaCl addition). ^b Expression 2 reflects the gene expression of the biofilm cells (NaCl addition). ^c log2 FC expresses the gene expression of the biofilm versus the planktonic cells. ^d A star marks a q-value (false discovery rate) lower than 0.001. Genes in bold are significantly differentially expressed (q-value < 0.001 and log2 FC <-1 or >1).</p

Biofilm Formation by <i>Clostridium ljungdahlii</i> Is Induced by Sodium Chloride Stress: Experimental Evaluation and Transcriptome Analysis

<div><p>The acetogen <i>Clostridium ljungdahlii</i> is capable of syngas fermentation and microbial electrosynthesis. Biofilm formation could benefit both these applications, but was not yet reported for <i>C</i>. <i>ljungdahlii</i>. Biofilm formation does not occur under standard growth conditions, but attachment or aggregation could be induced by different stresses. The strongest biofilm formation was observed with the addition of sodium chloride. After 3 days of incubation, the biomass volume attached to a plastic surface was 20 times higher with than without the addition of 200 mM NaCl to the medium. The addition of NaCl also resulted in biofilm formation on glass, graphite and glassy carbon, the latter two being often used electrode materials for microbial electrosynthesis. Biofilms were composed of extracellular proteins, polysaccharides, as well as DNA, while pilus-like appendages were observed with, but not without, the addition of NaCl. A transcriptome analysis comparing planktonic (no NaCl) and biofilm (NaCl addition) cells showed that <i>C</i>. <i>ljungdahlii</i> coped with the salt stress by the upregulation of the general stress response, Na⁺ export and osmoprotectant accumulation. A potential role for poly-N-acetylglucosamines and D-alanine in biofilm formation was found. Flagellar motility was downregulated, while putative type IV pili biosynthesis genes were not expressed. Moreover, the gene expression analysis suggested the involvement of the transcriptional regulators LexA, Spo0A and CcpA in stress response and biofilm formation. This study showed that NaCl addition might be a valuable strategy to induce biofilm formation by <i>C</i>. <i>ljungdahlii</i>, which can improve the efficacy of syngas fermentation and microbial electrosynthesis applications.</p></div

Distribution of up- and downregulated genes in the biofilm versus the planktonic condition over the different COG functional classes.

<p>Distribution of up- and downregulated genes in the biofilm versus the planktonic condition over the different COG functional classes.</p

Overview of the <i>C</i>. <i>ljungdahlii</i> phenotypes obtained in different stress conditions.

<p>The lower limit of the given concentration range reflects the lowest concentration at which the phenotype was observed, while at concentrations above the given upper limit, the induced stress was so strong that no growth was observed after ten days of incubation.</p

Investigation of the biofilm composition.

<p>A) Effect of enzyme treatment. <i>C</i>. <i>ljungdahlii</i> biofilms were grown in well plates by adding 200 mM NaCl to the medium. After 2 days of incubation, the supernatant of all wells was removed and the biofilms were washed twice with PBS buffer. PBS buffer without (control 1) or with 0.2 mg·mL^-1 proteinaseK or reaction buffer (10 mM Tris HCl pH 7.5, 2.5 mM MgCl₂, 0.5 mM CaCl₂) without (control 2) or with 2 U·mL^-1 DNaseI was added to the wells (n = 3). After one hour of enzyme treatment at 37°C, the crystal violet assay (described in text) was performed. B) Confocal laser scanning microscopy images of component-specific stained <i>C</i>. <i>ljungdahlii</i> biofilms. <i>C</i>. <i>ljungdahlii</i> was grown in chamber slides with the addition of 200 mM NaCl to the medium and, after 2 days of incubation, the biofilms were stained as described in the text with SYPRO ruby red biofilm matrix stain (left) or calcofluor white (right). The scale bars are 50 μm long.</p

Effect of the NaCl concentration on biofilm formation and growth.

<p>A) <i>C</i>. <i>ljungdahlii</i> was grown in well plates and NaCl was added to the medium in concentrations ranging from 0 to 280 mM (n = 3). The crystal violet assay (described in text) was performed after 3 days of incubation. The pictures underneath the data bars show the corresponding, stained biofilms, before extraction with methanol. Remark that OD 600 nm values are given on a linear scale, while the A 570 nm values are on a logarithmic scale. B) <i>C</i>. <i>ljungdahlii</i> was grown in tubes and NaCl was added to the medium in concentrations ranging from 0 to 280 mM (n = 3). Tubes were vortexed at a low speed to break up the formed aggregates, before measurement of the OD 600 nm.</p

Overview of the properties of the biofilms obtained with and without NaCl addition and on different materials.

<p>The maximum biofilm thickness was obtained using COMSTAT, while the surface coverage was determined using FIJI. Averages and standard deviations were calculated from values derived from at least three different images taken at different areas of the biofilm. The biomass was calculated from the maximum biofilm thickness and the surface coverage and is therefore also a maximum value.</p

TEM images of <i>C</i>. <i>ljungdahlii</i> appendages in different growth conditions.

<p><i>C</i>. <i>ljungdahlii</i> was grown in tubes without (left) or with (middle) the addition of 200 mM NaCl to the medium. In addition, <i>C</i>. <i>ljungdahlii</i> was grown with 200 mM sodium pyruvate, while fructose was omitted from the medium (right). After 2 days of incubation, cells were harvested and grids were prepared as described in the text. The scale bars are 500 nm.</p

Confocal laser scanning microscopy images of <i>C</i>. <i>ljungdahlii</i> biofilms.

<p>A) Cells were grown in chamber slides without (left) or with (right) the addition of 200 mM NaCl to the medium. B) Cells were grown in tubes, in which a piece of glass (left), graphite (middle) or glassy carbon (right) was placed vertically and to which 200 mM NaCl was added. After 2 days of incubation, the biofilms were stained with live/dead staining as described in the text. The scale bars are 50 μm.</p

Visual effect of different growth conditions.

<p><i>C</i>. <i>ljungdahlii</i> was grown in tubes without (left) or with (middle) the addition of 200 mM NaCl to the medium. In addition, <i>C</i>. <i>ljungdahlii</i> was grown with 200 mM sodium pyruvate, while fructose and was omitted from the medium (right). The pictures were taken after 3 days of incubation.</p