Wild yeasts are everywhere. Some of them will even make beer for you.

I spend a lot of time thinking about flowers and beer. Thinking about flowers is part of my job, and beer—that’s my current obsession. Thoughts collide, and I recently found myself dwelling upon what they have in common: that most marvellous microbe, yeast.

Yeast is that critical fungus that converts sugar solutions into beer and wine, and while we’ve got a handful of domesticated strains harnessed for beverage production, diverse and untamed wild yeasts are everywhere. They are in the air, on plants and animals, on your skin, in your hair. Wild yeasts are particularly abundant in flowers, and that’s because flowers provide a source of freely available sugar by way of nectar.

Saccharomyces_cerevisiae_SEM

Brewers/baker’s yeast (Source: Wikimedia Commons)

Given this obvious overlap, a natural and totally irresistible work-hobby collaboration sprang to mind. Could I capture a wild yeast from flowers of my study species and use it to make beer?

Now I am not the first to try this. Wild yeasts and other microbes have had a long history of use in creating beer. Belgian brewers have perhaps the most celebrated and storied traditions in this area—their Lambic beers are created by leaving fresh, unfermented beer (wort) to be inoculated by whatever yeast and bacteria the atmosphere may gift them. Wild fermentation is a growing global trend now, with numerous craft breweries here in Australia (e.g. La Sirene and Wildflower), and internationally (e.g. Allagash, Trinity) establishing strong reputations for artisanal ales fermented with the help of local microbial biodiversity.

While the diversity of wild yeasts might be wide, not all are useful for producing beer however. Many yeasts die in the presence of moderate alcohol, many cannot ferment all but the simplest of sugars, many produce unpalatable by-products during fermentation.

So where are we most likely to find the best, most useful wild yeasts for beer production? This is where floral biology meets brewing.

Floral biology meets beer brewing

Nectars are produced by flowers as rewards for the service of pollinating animals. Because some flowers specialize in being pollinated by particular kinds of animals, they evolve specific traits that cater to the biology of those animals. For example, moth-pollinated flowers are white so that they are visible in low light, bee-pollinated flowers evolve UV-reflective runway markers to guide accurate landing and foraging, carrion-fly pollinated flowers smell like rotting flesh. In the same fashion, nectar is shaped by evolution to cater to the specific creatures most likely to consume it.

One way nectar becomes tailored to its consumer is by its sugar concentration, which varies wildly. At the concentrated end, exceeding 50% sugar by weight, nectar is very viscous and sticky and difficult to suck up through long or thin mouth parts. These nectars cater to insects with short tongues like bees, flies, wasps and beetles. On the other end you have dilute nectars, with 10 – 25% sugar concentrations, and these are perfect for birds to lap up. By a happy coincidence, the sugar concentrations of bird-adapted nectars are in the same range as unfermented wort. Recognizing this was what led me to try hunting for yeasts in the flowers of my study species—the bird-pollinated shrub Prostanthera walteri.

img_2623

Monkey Mint-bush (Prostanthera walteri)

Also known as the Monkey Mint-bush, this is a rare shrub growing amongst boulders on a few misty, granite peaks in remote East Gippsland, Victoria. I have been getting to know the plant for a couple of years now, using it in a study to understand how bird-pollination might differ from insect-pollination. And so on a January field trip to collect some data, I took the opportunity to collect some fresh flowers and take them back to my home lab (kitchen bench) for bioprospecting. At home, I made up a test wort: a low concentration malt-extract solution to mimic the conditions of beer, then I syringed out the nectar from several flowers and spiked the test jars with whatever might be living in the nectar.

Screenshot 2019-05-14 14.50.19.png

Kitchen-bench inoculation of test wort

And it worked. Most of the test jars began fermentation, and sniffing the results revealed various aromas of bright apple juice, white wine, earth and smoke. After months of re-culturing these initial samples I now have what I think are two different strains* of nectar yeast, one of which just produced its first beer.

So how does it taste?

Interesting, and not bad… and that’s all I’m willing to venture at this stage of the experiment! The yeast fermented very quickly, and chewed through 79% of the available sugars (which is more than some domesticated brewing strains). It has a somewhat Belgian Saison-like character, with strong pear and floral esters, some smoke and spice, and a very slight tartness.

IMG_4219

I was quite blown away at how well this yeast performed, fermenting much like a domesticated yeast, yet with a much bigger, bolder, dare I say “wild” flavour. It is stunning to think that it has probably existed in flowers in remote eastern Victoria for some thousands to many thousands of years, and one can just go and pick it up and persuade it to make interesting beer. And as I get to know it better, perhaps that beer will become both interesting and delicious.

 

Thanks to Ruth Barry (Boatrocker Brewery) for inspiring conversation and advice on this.

*These are technically mixed cultures, but I believe they each have come to be dominated by single strain of yeast.

Unearthing diversity in fungal dark matter

To be born an orchid is a most unlikely thing. First your parents must be pollinated, which is difficult. Orchids are both rare, and rarely pollinated due to the bizarre and dishonest means by which they go about attracting pollinators. Added to that, orchids often rely on a single species of pollinator to do the job.

Let’s say, however, that your orchid parents do manage to achieve fertilization. Your orchid mother will produce many thousands of tiny dust-like seed, which will be jettisoned into the wind. Unlike most seeds, you have no maternal energy investment to power your germination and first days as a seedling. Instead, you must rely on blind luck to land you within reaching distance of a strand of soil fungus. This fungus is the wet nurse to bring you into the world, invading the seed coat and hooking the young orchid up to a network of fungal strands that pervade the soil. Tapping into this network provides you with the first sips of carbohydrate and nutrient you need in order to build your first green leaf and begin to stand on your own roots. But it is not enough to land near any fungus. Many orchid species require fungal partnership with a specific species of fungus for this to occur at all. Multiplied together, it is a wonder that orchids ever overcome these odds to propagate themselves into the next generation.

The southwest of Western Australia is rightly famous as a global biodiversity hotspot. The area is particularly rich in orchids, and the spider orchids (Caladenia) are some of the most impressive and diverse of the region’s main orchid groups. In 1967, University of Adelaide researcher John Warcup discovered in association with Caladenia a new genus of fungi. Today those fungi are called Serendipita, and although we have known of them for around 60 years, there have been less than a handful of species discovered and described.

img_0174

The spider orchid Caladenia arenicola was one of those sampled in the study

img_0494-1

White spider orchid (Caladenia splendens)

Ubiquitous yet invisible

Although related to mushrooms, Serendipita fungi have not been observed producing the conspicuous spore-bearing fruit bodies we usually use to find and identify them. This makes them largely invisible, and I have therefore never observed them in the wild. Despite that, recent research using DNA sequencing has found them to be absolutely everywhere. Inside all kinds of plants, outside all kinds of plants, and distributed from the equator to Antarctica. It is clear then that there must be a hidden biodiversity of these species siting, waiting to be discovered.

My study took a wide sample of southwest WA spider orchid samples and assayed them for the presence of Serendipita fungi. We then sequenced the DNA of all the fungi we found, and used a new analytical technique for dividing that DNA sequence diversity into units that are probably species. This is currently the only way to sensibly identify Serendipita fungi, as they all look completely alike and do not produce spores in the lab.

We found a total of eight species of Serendipita fungi, including the original species discovered by Warcup back in the 60s. These came from a total of 18 species of orchid. At some sites where we sampled multiple orchid species, we found six species of Serendipita, meaning that the fungi were as diverse as the orchids!

img_0970-1

Lying just below the soil horizon, that swollen, yellow stem bit is called the “collar”, and its where all spider orchids keep their fungus.

Untapped agricultural potential?

Although we have chosen to study these Serendipita in association with orchids, their wide host association has got other researchers interested in their role in plant health and application to agriculture. For example, Warcup’s species and one other have been used in experiments (and patent applications) showing inoculation with Serendipita results in profound benefits for the host plant, including:

  • Increased plant weight in maize, poplar, parsley, tobacco, barley, wheat, switchgrass and Arabidopsis
  • Enhanced grain yield in barley
  • Accelerated plant development in barley
  • Greater seed set, increased growth and faster flowering time in tobacco
  • Increased wheat yield in poor soils
  • Improved nutrient uptake in chickpea and lentil
  • Improved salinity tolerance in barley
  • Enhanced protection against root and stem pathogens in barley
  • Improved resistance to stem pathogens in tomato
  • Stronger defense response against mildew leaf pathogen in barley
  • Increased essential oil content in fennel and thyme
raycraven-sebacina-worldjmicro-2016

Figure 7 from Ray and Craven (2016): Root growth in winter wheat in Serendipita vermifera inoculated plants (left) versus control (right)

These proven benefits make Serendipita a potentially powerful tool to enhance plant productivity and stress tolerance in crops. Furthermore, application of Serendipita fungi could be an organic alternative permitting growers to lower the application of unsustainable and ecologically harmful synthetic fertilizers. Our knowledge of plant-Serendipita associations in the wild suggests that these relationships are more prevalent in nutrient poor soils such as those in southwest WA. They are probably one factor that allows our plant diversity to thrive in such weathered, poor soils. This means that species of fungi that have evolved with the nutrient poor soils (like those discovered in this paper) might be untapped tools to enhance agriculture taking place in those very same soils.

 

(Erratum: This story was edited to replace the figure attributed to Ray and Craven (2016). The first image I used was one showing Arabidopsis capability for mycorrhizal association. Arabidopsis is typically thought to be a non-mycorrhizal plant, which is why this is interesting. The image however showed slower growth in the mycorrhizal treatment. A related Serendipita has been shown to enhance root growth in Arabidopsis however. I have now updated the post with a more appropriate image of root growth gains in wheat. Thanks to Pawel Waryszak (@PWaryszak) for pointing this out.)

 

My study:

Whitehead, M. R., Catullo, R. A., Ruibal, M., Dixon, K. W., Peakall, R., & Linde, C. C. (2017). Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity. Fungal Ecology, 26, 74-84.

Further reading:

Weiß, M., Sýkorová, Z., Garnica, S., Riess, K., Martos, F., Krause, C., … & Redecker, D. (2011). Sebacinales everywhere: previously overlooked ubiquitous fungal endophytes. Plos one, 6(2), e16793.

Weiß, M., Waller, F., Zuccaro, A., & Selosse, M. A. (2016). Sebacinales–one thousand and one interactions with land plants. New Phytologist, 211(1), 20-40.

Ray, P., & Craven, K. D. (2016). Sebacinavermifera: a unique root symbiont with vast agronomic potential. World Journal of Microbiology and Biotechnology, 32(1), 16.

Bokati, D., & Craven, K. D. (2016). The cryptic Sebacinales: An obscure but ubiquitous group of root symbionts comes to light. Fungal Ecology, 22, 115-119.