How do we get the microorganisms from your sourdough samples?
There are a lot of microscopically small organisms in sourdough. Knowing which ones tells us a lot about the characteristics of the sourdough, as each microbial species exhibits a particular, characteristic metabolism. For example, some yeast species produce a lot of carbon dioxide (CO2), while others produce barely any, affecting the leavening capacity of the sourdough. Lactic acid bacteria produce lactic acid (as their name suggests), but different species produce varying amounts, leading to a more or less acidic taste. Many other compounds are produced in different amounts by different microorganisms.
As such, we want to know in detail which microorganisms are present in the sourdough samples we receive. For that, multiple methods are being used. The most work-intensive method involves selective plating followed by identification. This is the method we will address in this blog post. There is a second method that does not rely on selective plating but is purely DNA-based, for which advanced equipment like the KingFisher is being used to extract the DNA, which we describe in this blog post. Some collecting partners in the HealthFerm project use both techniques to account for the shortcomings of each, while others only use one.
How do we know what to look for?
As with humans, where different people and cultures like to eat different types of food, micro-organisms are similar, in that they prefer certain media (types of food, that is) but they are a lot pickier. Some species do not even grow when the medium is not optimal. Thus, we need to know what types of microorganisms we are looking for and what they require to grow.
We know from previous research that a sourdough typically contains a large amount of lactic acid bacteria, accompanied by one or two yeast species. In about 30 % of the sourdoughs, acetic acid bacteria are present as well. Hence, we work with five different growth media to make sure we can grow all the microorganisms present.
With this knowledge, we can go to the laboratory to cultivate the sourdough microorganisms and reveal their presence to us!
Let’s say a sourdough sampling kit just arrived in our laboratory through the postal service. Inside, we find the sampling pot that was filled by the citizen scientist. From the sample, we take a few grams for microbial analysis and dilute it with physiological water, a 0.9 salt solution, to ensure the sourdough suspends well. This resulting watery mixture contains an immense amount of microbial cells, anywhere between 10 million to 1 billion cells per milliliter.
©Vrije Universiteit Brussel
To allow these cells to grow in separate colonies on plates with agar media, we make dilutions of that watery phase. On these plates, the bacterial and yeast cells can take up all the nutrients they require to grow and multiply. After two to ten days, most microorganisms have grown into colonies visible to the human eye. Typically, we obtain between 30 and 300 colonies if we choose the dilutions correctly. About 10 % of these colonies are randomly selected and further grown and analyzed in test tubes.
©Vrije Universiteit Brussel
This way, our ultralow temperature freezers quickly get filled with microbial isolates from all the sourdough samples we receive. But we do not know anything about them yet!
What we would like to know first is the species they belong to. This allows us to dive into the scientific literature to learn what the role of these microorganisms is in sourdough.
Under usual circumstances, finding out the species to which an organism belongs can be done either by looking at them or by examining their DNA (the blueprint or code of an organism). To know whether Dolly is a cow or a sheep, it is as simple as looking at Dolly and identifying typical features. However, since microorganisms cannot be easily distinguished by the naked eye, we have to examine their DNA.
To do so, we must first extract the DNA from the cells. We use a special protocol to open the bacteria and yeasts to get access to the cells’ internal content. At this stage, we could determine the nucleotide sequence of the full DNA, also known as the genome, for each of the thousands of isolates we obtain from the sourdough samples. As this would be time and resource-intensive, we simplify this task by focusing on only a few genes (pieces of the code responsible for specific features) present in all microorganisms that are sufficiently different in nucleotide sequence to distinguish between different species. Using laboratory techniques like polymerase chain reaction (PCR), we make more copies of these genes to easily determine their sequence. We then compare these sequences with databases containing validated information to identify our microorganisms.
After all this work, we have a vast amount of microorganisms identified. All that is left is to dive into this data, determine the microorganisms that co-occur together, which are most abundant in each sourdough, and which characteristics of the sourdoughs can be related to the microorganisms.
