A giant cell of the fungal pathogen Cryptococcus neoformans. Credit: PLoS Pathogens Issue Image
Fungi cover a broad range of organisms, from mushrooms to moulds, to yeasts, and they are extremely good at breaking down all sorts of things. Man-made materials like dyes can be difficult to break down as they aren’t naturally occurring, however there have been fungi found which can break down plastics, rocks, and iron, and so it is no surprise that they are some of the most effective microorganisms for degrading dyes as well.
Black moulds are probably the most common fungi. Credit: Pinterest
Fungi can break down dyes in a few different ways, so depending on the kind, you could expect to see biodegradation, biosorption, azo bond reduction or bioaccumulation. Fungi can also create enzymes such as laccase, azoreductase, and peroxidase to help with breaking down dyes. Of course, these enzymes and fungi work best under specific conditions, and to stop these enzymes from denaturing they generally need to be in slightly acidic conditions, and temperatures of around 30°C.
Of all the fungi capable of breaking down dyes, some stand out for how versatile they are, or just how commonplace! Some examples of very common fungi which are able to break down dyes are bakers’ yeast (Saccharomyces cerevisiae), white rot fungi (Pyricularia oryzae) and black mould (Aspergillus niger - often found on your rotten tomatoes). Because many of these fungi produce powerful enzymes, they can break down not only the original dyes but also any intermediate chemicals the dyes turn into. This is very important, as many of these intermediate chemicals are very hazardous to human health. Unfortunately, fungi also take quite a long time to do this compared with other microorganisms.
Saccharomyces cerevisiae (Baker's yeast) Credit: National Collection of Yeast Cultures (NCYC)
Conidia of P. oryzae with germ tubes and appressoria Credit: R. J. Howard
Aspergillus niger Credit: PaxDb
Preparing yeast mixtures in the Lab.
One experiment which can show the effect that yeast has on dyes is simple enough that it can be done at home. By mixing dye, water, sugar and yeast together and leaving the yeast to activate, there will be a gradual colour change. In the case of our mixture, the colour went from purple-black to red after one day of activity. This may be because the blue and black dyes were easier to break down than the red ones. When we did the same experiment with almost no sugar there was far less colour change. Yeasts usually decolour by biosorption, taking the dye into their own cells, rather than by using enzymes. That means you can see a colour change in the yeast as well as the water at the end of the experiment. In a mixture of dyes, this leaves a banded pattern, as some of the dyes are absorbed faster than others. Because of this, each settling layer of yeast will have a different set of chemicals to draw colour from and will have a different hue.
Ink, Baker's yeast and sugar water mixture after 0hr, 24hr, 48hr. The decolourisation can be observed by naked eyes.
It is possible to replicate the effect fungi have on dyes by looking at what happens when the dyes are exposed to enzymes. As many kinds of fungi break down dyes by producing enzymes, doing experiments using enzymes is a quicker way of estimating what kind of effect the microorganism itself would have on it. In this case, we’d expect to see fungi able to produce laccase, azoreductase, and peroxidase, which have been shown to be able to break down dyes. By mixing our dye with these enzymes (as well as pectinase, viscozyme L, and alcalase) and waiting a few days, it was possible to see that the colour becomes very faded in the samples where enzymes were added compared to the samples where no enzymes were added.
Ink samples with laccase, lysozyme and yeasts mixtures showed obvious decolourisation results.
Agar plates pre-mixed with 0.03% (left) and 0.15% (right) dye concentration
One final method of testing a fungi’s ability to break down dyes is through their cultivation. For this experiment, a nutrient-rich Luria broth was made, with 0.03% and 0.15 % dye. After a series of agar plates with both these concentrations were made, they were exposed to microbes from various areas and left to grow for three days. While it wasn’t possible to find out exactly what was growing on the plates, we were able to make assumptions based on where the microbes originated. One of the best microbial samples for decolouring dyes, for example, was taken from a batch of rotten tomatoes which usually contain Aspergillus niger. There is already a body of evidence that Aspergillus niger can degrade inks, so seeing such good decolourisation suggests its presence on the agar plates.
One of agar plates that cultivated with Aspergillus niger showed some partial decolourisation result (top right area).