Resistance to molds of flax-fiber based materials

In the present study, the prepared covering material based on flax fibers containing caffeine, nano-copper, or corundum (Al2O3) as a potential biocidal substance was prepared and tested on biological resistance. Two experimental designs were performed: a) the samples were placed in a desiccator at the air humidity of about 90 % and at a temperature of 25 °C for one month. The samples were then transferred to sterile agar and the coverage of the agars and the samples with molds were observed after a week of incubation. All samples were found to be infected. The samples containing copper were the least resistant. b) The samples were placed on sterile agar and the petri dishes were subjected to the fallout method, where both the samples and the agars were insulted by mold spores from the air. The Petri dishes with the samples were then placed in a thermostat in the dark at 25 °C for a week. After the end of the exposure, the samples and agars were observed. The samples were covered by molds with an increasing intensity in the following rank: caffeine sample = corundum sample < Cu sample = control sample = pure flax fibers.


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].One of such straw/s material is linen.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.For now, however, the flax-based material has not been tested in a mixture with other substances with biocidal effects.Caffeine (1,3,7trimethyl-3,7-dihydro-1H-purin-2,6-dion) belongs to the biocidal substances, its effects against the growth of wood-decaying fungi and molds was observed) [5][6][7][8][9][10].On the other hand, this substance is consumed in drinks and food, drugs, or cosmetics by millions of people each day in the whole world without any problems with healthy if it is used at normal levels.The more known antifungal element is a copper.Its antifungal properties were confirmed, especially when copper was applied in a nanoform [11][12][13][14][15].The other possibility is an addition of corundum (Al2O3).It is used for surface roughening.Corundum has not ever been studied as an antifungal reagent for building materials production.
In the present study, bio-component based on flax-fibers without antifungal additives or with two concentrations of caffeine or nano-copper or corundum were studied in two following experiments: a) under defined laboratory conditions in a desiccator and the highest optimal air humidity for molds (95 %) with verification with the verification of molds on the cover on the samples on the agar and b) on the agar medium by a fall-up method when sterile samples and sterile agars were covered by molds from the air.

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 [5][6][7][8][9] or according to experience with the preparation of studied composite flax materials (Brejcha, personal communication).
Samples of bio-composite panels were made with 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 of clear glass.

Manufacturing process
The surface was polished and then treated by a 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.The system was settled to the vacuum level.Subsequently, the system was left for 30 minutes to check the tightness of the vacuum bag.The 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 at room temperature at 22 °C for the next 24 hours.

Sample identification
The labelling of the samples and their number in the experiment A or in the experiment B are shown in table 1.
Table 1.The samples and their labelling in the experiment A and B and numbers of their replicates.

Experimental design of tests with molds
Experiment A) The prepared samples were placed in a desiccator at the air humidity of about 90 % and at a temperature of 25 °C for one month (figure 1).The correct air humidity in the desiccators was made by K2SO4 aquatic solution under the frontier of solubility in water.The samples were then transferred to sterile agar and the coverage of the agars and the samples with molds were observed and photographed after a week of incubation under a temperature of 25 °C.Experiment B) The sterile samples (flax-fiber sample containing caffeine (1 or 2 %), nano-copper (2 or 4 %), corundum (2 or 4 %), and pure flaxfiber) were placed on sterile agar and the Petri dishes were subjected to the fallout method, where both the samples and the agars were insulted by mold spores from the air.Three replicates were made for each variant.The Petri dishes with the samples were then placed in a thermostat in the dark at 25 °C for 168 hours.After the end of the exposure, the samples and the agars were observed and photographed.

Results and discussion
The experiment A) The samples taken out of the desiccator, were not covered on the first view by molds, and the quantity of potential fungal spores could not be meaningfully evaluated even under a binocular microscope.For this reason, the samples were placed on sterile maltose agar (5%) and then the growth of fungi was observed in the dishes.The fungi in all dishes covered not only their own samples, but also agar, which was a source of nutrients for them.Controls, samples with caffeine, and samples with corundum were covered by molds less, the molds covered more agar and glass of dishes than own samples.The samples containing copper were the least resistant (see figure 2), they were also covered by molds from the bottom.It was probably caused by the higher wettability of samples with copper in comparison to samples with caffeine or corundum (M.Böhm and V. Brejcha, personal communication, unpublished results).In every case, any of the samples was not able to completely resist the action of molds.The experiment B) All samples were in a contact with molds, with the exception of sterile control agar without any samples.The studied samples were covered by molds with an increasing intensity partially graded in the following rank: caffeine sample = corundum sample < Cu sample = control sample = pure flax fibers (see figure 3).Growth of mycelium on the surface of the samples was individually rated (see figure 4) using a five-degree scale with 0 indicating no growth and 5 indicating heavy mold growth [20].The use of flax for construction purposes has recently been addressed by several studies [16][17][18][19].However, their results cannot be completely compared, because they were carried out under different conditions.In any case, it can be summarized that flax-based materials are very sensitive to biodegradation, and various additional biocidal substances that could correct this are not yet completely effective.There is a fact that all the studies have been carried out at high humidity, when mold thrives well as in our experiment A. However, our experiment with agar and airborne mold spores suggests that these materials are unfortunately likely to be attacked over time even in less humid conditions and it cannot be ruled out that these samples would be protected under normal humidity (40-60% air humidity).
Effectivity of caffeine against molds has been confirmed in many studies in the case of wood materials [5,9] and the same conclusion can be described for cuprum generally.Corundum has never been tested for biocidal use according to the available literature.The component causing its toxicity is aluminium.Aluminium is not a natural and biogenic element for organisms, so its biocidal effect is not surprising on the other hand.In the future, so it would be advisable to test durability of our flax-fiber material also in normal humidity in practise or in some long-term experiments.It will also probably be necessary to pre-treat the flax fibers or the entire manufactured bio-composite in some way in order to reduce their biological attack.

Conclusions
In this study, caffeine, nano-copper or corundum were added into the flax-based material for surface applications as potential biocidal substances.The samples were exposed to 90% humidity (as the worst case of exposition scenario), which provides optimal moisture conditions for mold growth.The ability of samples to resist mold growth was then determined.Another method was an experiment with airborne fungi on a nutrient agar medium.In both cases, all samples, whether with or without a biocidal substance, were attacked by molds.The most interesting combination seems to be a flax material containing caffeine, but further research will be needed to verify its applicability (lower concentration than 1% of caffeine and various applications on flax material).

Figure 1 .
Figure 1.Samples in a desiccator (the experiment A).

Figure 2 .
Figure 2. The experiment with the samples from the previous placement in a desiccator.The Petri dishes are photographed from the top (the picture on the left) and from the bottom (the picture on the right).Dish labels: C -control samples (prepared sample without an additive or fabric, K -samples with caffeine (1 or 2 %), Cu = samples with copper (2 or 4 %), Al2O3 = samples with corundum (2 or 4 %).

Figure 3 .
Figure 3.The samples on agars from the experiment with aerial unspecific mould community.The Petri dishes are photographed from the top (picture on the left) and from the bottom (picture on the right).Dish labels: Co = control clear agars, R = flax-fiber samples without an additive, C 1 % and C 2 % = samples with caffeine, Cu 2 % and Cu 4 % = samples with copper, Al 2 % and Al 4 % = samples with corundum (from left to right in both photos).3 replicates were made for each the variant.

Figure 4 .
Figure 4.The rating scale of mold growth according to the [20].