Influence of Different Nanomaterials on Growth and Mycotoxin Production of Penicillium verrucosum.

Nanoparticles are ubiquitous in the environment. They originate from anthropogenic or natural sources or they are intentionally produced for different purposes. There exist manifold applications of nanoparticles in modern life leading unavoidably to a confrontation and interaction between nanomaterial and living organisms. Based on their wide distribution tending to increase steadily, the influence of particles based on silica and silver, exhibiting nominal sizes between 0.65 nm and 200 nm, on the physiology of the mycotoxigenic filamentous fungus Penicillium verrucosum was analyzed. The applied concentration and time-point, the size and the chemical composition of the particles was shown to have a strong influence on growth and mycotoxin biosynthesis. On microscopic scale it could be shown that silver nanoparticles attach to the mycelial surface. Moreover, silver nanoparticles with 0.65 nm and 5 nm in size were shown to internalize within the cell, form agglomerates in the cytoplasm and associate to cell organelles

Mycotoxin biosynthesis and intracellular ROS level of <i>P</i>. <i>verrucosum</i> supplemented by NanoComposix SiO₂ nanospheres.

<p>ROS and mycotoxins have been analyzed at the same time points. (<b>a</b>) Biosynthesis of citrinin and ochratoxin A by <i>P</i>. <i>verrucosum</i> growing 7 days at 25°C under shaking at 230 rpm in MEA-liquid medium supplemented by different amounts (0–2,500 ppm) of NanoComposix SiO₂ nanospheres exhibiting sizes of 20 nm, 50 nm, 100 nm and 200 nm. (<b>b</b>) Determination of intracellular ROS in <i>P</i>. <i>verrucosum</i> at the end of 7 days of growth at 25°C, under shaking at 230 rpm, in MEA-liquid medium, supplemented by different amounts (0–2,500 ppm) of NanoComposix 50 nm sized SiO₂ nanospheres. Logarithmic calculation. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150855#pone.0150855.s003" target="_blank">S3 data</a> set.</p

Specifications of the applied nanomaterials (Data in S5 data set).

<p>Specifications of the applied nanomaterials (Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150855#pone.0150855.s005" target="_blank">S5 data</a> set).</p

Stability of the nanoparticles in MEA (liquid) medium over 7 days.

<p>(a + b) NanoComposix silica (50 nm, 1,000 ppm). (c + d) NanoComposix silver (50 nm, 10 ppm). (e + f) Purest Colloids MesoSilver^® (10 ppm). Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150855#pone.0150855.s001" target="_blank">S1 data</a> set.</p

Relative growth density reflecting the growth rates and mycotoxin biosynthesis of <i>P</i>. <i>verrucosum</i> supplemented with sub-nano sized MesoSilver (0.65 nm).

<p>(<b>a</b>) Growth rate of <i>P</i>. <i>verrucosum</i> BFE575 at different concentrations of sub-nano sized MesoSilver (0.65 nm) 7 days at 25°C under rotation of 230 rpm in MEA-liquid medium. (<b>b</b>) Growth and mycotoxin biosynthesis visualized by TLC of <i>P</i>. <i>verrucosum</i> growing 7 days at 25°C under rotation of 230 rpm in MEA-liquid medium supplemented by different amounts of sub-nano sized MesoSilver (0.65 nm). MesoSilver has been applied <b>before</b> (!) growth of <i>P</i>. <i>verrucosum</i> (<b>c</b>). Growth and mycotoxin biosynthesis visualized by TLC of <i>P</i>. <i>verrucosum</i> growing 7 days at 25°C under rotation of 230 rpm in MEA-liquid medium supplemented by different amounts of sub-nano sized MesoSilver (0.65 nm). MesoSilver has been applied <b>after</b> (!) growth of <i>P</i>. <i>verrucosum</i>. Significant differences (P < 0.001) are indicated with an asterisk. The growth rate is specified as relative growth density. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150855#pone.0150855.s004" target="_blank">S4 data</a> set.</p

Relative growth density reflecting the growth rates of <i>Penicillium verrucosum</i> supplemented by NanoComposix SiO₂ nanospheres.

<p><i>Penicillium verrucosum</i> incubated 7 days at 25°C under shaking of 230 rpm in MEA-liquid medium, supplemented by different amounts (0–2,500 ppm) of NanoComposix SiO₂ nanospheres exhibiting sizes of 20 nm, 50 nm, 100 nm and 200 nm. Significant differences (P < 0.05) are indicated with an asterisk. The growth rate is specified as relative growth density. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150855#pone.0150855.s002" target="_blank">S2 data</a> set.</p

Microscopical examination of <i>P</i>. <i>verrucosum;</i> grown with 5 nm NanoComposix silver nanospheres or MesoSilver with 0.65 nm.

<p>Incubation was for 7 days at 25°C under rotation of 230 rpm. Magnification was 1000x using oil-immersion. Red marks show agglomerations of silver nanoparticles outside the cell and within the mycelial filaments in the cytoplasm and partly on the surface of nuclei and other organelles.</p

Growth morphology of <i>Penicillium verrucosum</i> supplemented by NanoComposix silver nanospheres.

<p>(a) Growth of <i>Penicillium verrucosum</i> 7 days at 25°C under rotation of 230 rpm in MEA-liquid medium supplemented by different amounts (0–100 ppm) of NanoComposix <b>silver</b> nanospheres exhibiting sizes of 5 nm, 50 nm and 200 nm. Silver has been applied <b>before</b> (!) growth of <i>P</i>. <i>verrucosum</i> (b). Growth of <i>Penicillium verrucosum</i> 7 days at 25°C under rotation of 230 rpm in MEA-liquid medium either as control or supplemented by different amounts (0–100 ppm) of NanoComposix <b>silver</b> nanospheres exhibiting a size of 200 nm. Silver has been applied <b>after</b> (!) growth of <i>P</i>. <i>verrucosum</i>.</p