Viability of yeasts after exposure to flax-fiber based composite materials

In the present study, the prepared bio-composite materials based on flax fibers containing caffeine or nano-copper (Cu) or corundum (Al2O3) as a potential biocidal substance were prepared and tested on their toxicity. Two viability tests were performed. Firstly, the screening test based on colouring by Methyl-blue was applied on yeast cells of strain Saccharomyces cerevisae. The number of cells density was observed under microscope and Methyl-blue was then added. Part of the cells coloured in blue and it indicated their death. For this reason, MTT viability assay was performed. The method is based on production of blue product formazan by yeasts after their metabolization of added initial substrate, 3-[4,5-dimethylthiazol-2-yl]-2,5-difenyltetrazolium bromide. The intensity of blue-coloured formazan was measured spectrophotometrically at 485 nm. The results indicated that the inhibition of yeasts was increased in the following rate: cu-samples (14-16 %) < flax sample without biocidal substances (21%) = pure flax (23 %) < caffeine-samples (up to 29 %) < corundum-samples (up to 35 %).


Introduction
In recent years, the use of natural matrices of plant origin in building materials has been growing (kenaf, wood, hemp, coconut, cane and straw) [1][2][3][4][5][6][7].One of such straw material is a flax.This plant is commonly grown in Central European conditions and is also relatively cheap and available.Its use for insulation purposes has already been described in several studies.However, such materials are organic origin and they can they can easily undergo biological degradation.For this reason, it is necessary to add biocidal substances to innovative building materials on a natural basis.These can also be of natural origin, but they are often various metals, organic persistent substances that can be toxic to the environment or humans.For this reason, it is also necessary to subject newly created materials to toxicological analyses.
One of the most commonly used organisms is yeast.Structurally, its cells are the transition between bacteria and cells found in the organism of plants, animals and humans.Yeasts are mainly used in tests where the biomass of cells or the viability of cells is evaluated after exposure to the sample.Biomass can be calculated thanks to microscopical observation or to spectrometrically at a specific wavelength [8].Viability is analysed by many methods, e.g.indirectly by cultivation on agar or directly by colouring of death cells by methylene blue [9] or propidium iodide [10] or producing of some specific product of metabolism (MTT test) or by flow cytometer [11].Principle of assay with the methylene-blue is based on a fact that only death cells are coloured blue, living cells do not pass the dye into the cell.Staining can be observed under a microscope to express the percentage of stained cells.It's a fast, but not very accurate method because it's time-depending.After a certain time, all the cells release the dye.Therefore, other methods have been invented, which provide more information about the metabolic processes taking place in the cells and are not so demanding on the duration of observation and evaluation.Test based on enzymatic activity of cells (MTT assay) belong to such more informative method.It is based on presumption that healthy cells metabolise added substrate and produce any specific coloured product and its amount is measured by spectrophotometer.
In the present study, toxicity of bio-composite based on flax-fibers containing caffeine (organic substance with verified biocidal effects) or nano-copper (verified antimicrobial effects) or corundum (Al2O3, when aluminium is toxic, non-essential element) as potential biocidal additives were compared in two above introduced viability tests (methyl blue assay and MTT assay) with yeasts species Saccharomyces cerevisae under defined laboratory conditions.

Organisms
Yeasts (strain Saccharomyces cerevisae) were donated by the Research Institute of Brewing and Malting (Prague, Czech Republic).

Chemicals
Caffeine was purchased from the company Sigma Aldrich, Ltd. (Czech Republic), nano-copper (99.9% purity) from EM-Tec (Germany), and white corundum (<100um particle size, 99.7% Al2O3) from PKIT (Czech Republic).All samples were in the form of powder.Deionized water was used as a solvent for the preparation of aquatic solutions with all three additives.Concentrations of caffeine were 10 g or 20 g/L, copper and corundum concentrations were 20 or 40 g/L.These concentrations were selected on the basis of previous toxicity data with molds and fungi [12][13] or according to experience with the preparation of studied composite flax materials (Brejcha, personal communication).
Methyl-blue powder was purchased from the company P-LAB, Ltd. (Czech Republic).The crystals of color were soluble in distilled water.Concentration 0.1 g/L was used.
Samples of bio composite panel where made by flax fiber cloth (2 layers of 200g/m 2 , Flaxdry BL200, EcoTechnilin) and epoxy resin (IB2 Epoxy Infusion Bio Resin, Easy composites).The theoretical weight ratio of natural fibers and resin was set to 1:1.Infusion technology was chosen for the production process.The base surface was made by clear glass.

Manufacturing process
The surface was polished and then treated by separation system (CR-1 Easy lease Chemical Release agent, Easy Composites).After 2 hours it was followed by the application of two layers of flax fiber cloth in a dry form and its closure into a system of auxiliary foils and a vacuum bag.System was settled to the vacuum level.Subsequently, the system was left for 30 minutes to check tightness of the vacuum bag.Next step was to prepare a mixture of epoxy resin in a weight ratio of 100:22.After thorough mixing, the resin was sucked into the fabric.The process of infusion of additives took approximately 8 minutes in total.The composite was left in room temperature 22 °C for next 24 hours.The flax-epoxy samples (without or with copper, corundum or caffeine as additives) were cut on squares about the size 1 x 1 cm and used for chemical or biological analysis (see figure 1).

Preparation of eluate from flax-based samples
Each of the prepared materials was used for a making of eluate in a volume ration 1:1.The mix of material and distilled water was shaken on a shaker REAX in a vertical position for 24 hours.Then the sample squares were passed through a sieve and the pure leachate was used for chemical and biological analyses.

Description of caffeine analysis
Caffeine residues in aquatic eluates were analyzed by UV-VIS spectrometry at wavelength 287 nm according to [14].Pure distilled water (as a background), the eluate from material without presence of caffeine, the eluates from materials with caffeine (10 or 20 g/L) were tested.

Description of metals analysis
Prior to metals analysis of the eluates (with copper and corundum and controlsflax, composite material) were stabilized by nitric acid (0.1 ml of acid into 100 ml of sample) and kept refrigerated.For analysis of copper (Cu) and aluminium (Al) atomic absorption spectrometry with electrothermic atomization (AAS iCE 3500Z Thermo Scientific) was used.

Experimental design of tests with yeasts 2.7.1 Viability test with methyl-blue
10 ml of eluate containing yeasts of S. cerevisae (10,000 cells/ml) were incubated in glass flasks and covered by plastic lids at the temperature 35 °C in a dark for 24 hours.Three replicates were used for each of the sample.10 µl of solution was dropped onto a glass slide, covered with a coverslip, number of cells in five volumetrically defined fields were estimated for each the sample and control.After that, methyl-blue dye was added.The dye was left to act for 2 minutes and the ratio of stained cells to unstained cells was observed under a light microscope in the same five fields (figure 2).
]-2,5-difenyltetrazolium bromide was performed in plastic tubes with lids. 10 ml of puffer with yeasts S. cerevisae (10,000 cells/ml) were incubated at the temperature 35 °C in a dark for 24 hours.2 replicates with yeasts and one replicate without organisms as a control were prepared for each of the samples.After the period, 0.5 ml of 3-[4,5-dimethylthiazol-2-yl]-2,5-difenyltetrazolium bromide (5 g/L) was added into each of the tube.The samples were incubated at the temperature 35 °C in a dark for the next 24 hours.The next day, 5 ml of ethanol was mixed with a contain each of the tube.The tubes with ethanol were left for next 24 hours in a dark.The coloured blue solutions were measured by spectrometer at wavelength 485 nm.The absorbance values were overestimated on inhibition in comparison to controlflax-composite without of any biocidal substance and control culture from nutrient solution (figure 3).

Calculations
Inhibition of absorbance (A) values (relevant for the subchapter 2.7.2) was calculated according to the equation ( 1): where Acontrol = mean absorbance value for control sample, Asample = mean absorbance value for the specific sample.The results of chemical analysis are described in the table 1.It is evident that distilled water contained very low background levels of caffeine or copper.Aluminium was not detected in controls as well in the tested samples of bio-composite containing Al2O3.It is not surprising, because aluminium in this form should be insoluble in the water and that is so is not detected in the leachates.On the other hand, some residues of caffeine or copper were analysed in the eluates from tested building samples.Caffeine residue were negligible.Copper was the most leachable biocidal component of the all.Yet everything, its residues were relatively low.

Viability test with methyl blue
Number of live or death yeasts was observed under a microscope in Bürker counting chamber.The ration of died (blue) yeasts in contrary to live (colorless) are presented in percent in the figure 4. Its to see that all samples contained dead cells.Their ration was increased in the samples with copper (more than 25 %) and corundum (more than 50 %).This method is not, however, very precise, because the addition of dye under the coverslip always more or less leads to the displacement of cells to other surrounding fields.The calculation of live versus dead cells is therefore never completely definitive and should only be rather taken as a screening indicator.In every case, the results of the method showed that some of the cells are not in a good condition and this indicates the effect of the studied samples on their health (possible damage of cell membranes, oxidative stress or the other possible effects).For the reason, the test based on enzymatic activity was additionally performed (the subchapter 3.3).

Viability test with 3-[4,5-dimethylthiazol-2-yl]-2,5-difenyltetrazolium bromide
This test is based on the determination of the formazan substance in the samples.Formazan is created as a product of the metabolism of the present microorganisms, in our case the yeast species S. cerevisae.
Only living yeasts are able to use the supplied substance 3-[4,5-dimethylthiazol-2-yl]-2,5difenyltetrazolium bromide for their metabolic processes and use it as a source of nutrients.The result of their metabolism is just the formazan, which is purple colored.Therefore, the higher the absorbance value measured in the test, the higher the amount of formazan was in the test tube and this leads to the conclusion that the more cells are alive.The measured absorbance values are described in the table 2.
The results indicate the highest metabolic activity at control (yeasts without samples) or in the eluates with caffeine (1%) or copper (2 and 4%s).Surprisingly, pure flax and composite based only on flax fibers had lower absorbance values.The worst results were found for caffeine (2 %) and corundum (both the concentrations).It is possible the yeasts did not have many nutrients in the samples without biocidal additives.It is known that copper is an important bio-element that also participates in the metabolic activities of cells [e.g., 15].This element could therefore paradoxically support yeasts growth and metabolic activity.Only higher concentrations of caffeine and samples with corundum showed increased cell toxicity of around 30% in comparison to flax and composite material (compare data in the table 2).
From toxicological point of view, corundum samples such seems to be the most toxic followed by samples with caffeine.The material containing copper or material with the lower tested caffeine concentration were less danger than the basic material without any additive substance.Unfortunately, it is not possible to compare our results with the literature, as a similar study has not yet been published.For the reason, we can recommendedn rather production and use of our flax based composite with addition of 1% of caffeine thanks its low toxicity and leachability on the one hand and for its lower biodegradation potential than for our developed flax material with copper on the other hand (personal data not yet published).Table 2. Absorbance values of the control nutrient medium, and the eluates of the flax-composite without biocidal substances, of the samples containing caffeine (Cf), of the samples containing nanocopper (Cu) and of the samples containing corundum (Al).Absorbances were analysed on the spectrophotometer at 485 nm.

Figure 2 .
Figure 2.An example of death blue cells and live non-coloured yeasts from the experiment.

Figure 3 .
Figure 3. Photo of the results of MTT assay -the violet colour indicates presence of measurable product formazan, the yellow solutions are samples without produced formazan.The slimmer tube in the right contains yellow solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-difenyltetrazolium bromide.

Table 1 .
Caffeine (Cf), copper (Cu) or aluminium (Al) concentrations in the eluates from the samples containing caffeine, nano-copper or corundum and in the distilled water.Caffeine was analysed on the spectrophotometer at 287 nm and both the metals by AAS.