Microbial co-culture part two: The science.

A while back, I wrote a post about the concepts that underpin the benefits of microbial co-culture with a promise to re-visit the topic and focus more on the science behind fermentation. Here it is!

Fermentation is one of humanity’s oldest biotechnologies. From the complex flavors in our favorite cheeses to the frothy pleasure of beer, fermented products have shaped our diets and cultures for millennia. But fermentation science extends far beyond food and drink. It underpins modern biotechnology, from pharmaceuticals to biofuels, to, of course, one of our favorite pursuits here at Impello: making co-fermented microbial products for agriculture. 

Understanding and consistently controlling fermentation—whether in a traditional cheese cave or a cutting-edge bioreactor—requires careful management of microbial life. And as the science around growing microbes advances, fermentation is evolving beyond single-species (monoculture) systems to more complex and innovative co-culture fermentations which require yet more nuanced caretaking and supervision of microbes. 

The oldest cultures are co-cultures: Fermentation in everyday life.

Precision co-culturing might be new, but the oldest human-manipulated fermentations are also co-cultures. Most people are familiar with fermentation through their favorite foods and beverages. Cheese, for example, relies on lactic acid bacteria (LAB) to acidify milk, followed by fungal or bacterial cultures that go on to create distinct flavors and textures. Beer is another well-loved fermented product, where yeast (very often Saccharomyces cerevisiae) consumes sugars from malted grains to produce alcohol and carbon dioxide (the bubbles!) Similarly, sourdough bread gets its airy rise and signature taste from wild yeasts and LAB living in symbiosis (an ecological partnership). And, agriculture is no stranger to the benefits of these complex communities either, humans have been fermenting and using compost and its beneficial microbes for millennia.

While these traditional fermentations are technically complex co-cultures, they are largely consistent and successful because they use well-understood microbes and made via practices that have been refined over centuries. Just like in any healthy agricultural system, the “health” of a co-culture lies in the diversity of its organisms which enable it to stay in balance. But what happens when fermentation moves beyond the artisanal and into industrial biotechnology?

Monoculture fermentation: Precision at scale

In most industrial settings, fermentation has historically relied on monocultures—single microbial species grown under tightly controlled conditions to maximize production of a desired product. These products are “metabolites” or desirable molecules that are produced as a byproduct of microbial metabolism. This approach is used to manufacture things such as antibiotics, bioethanol, and even synthetic food ingredients like citric acid, among many other uses.

To achieve efficient monoculture fermentation, scientists and engineers monitor several key parameters:

The pH: If you remember your basic chemistry, pH is a measure of how acidic or alkaline a solution is. This is important because most microbes thrive within a specific pH range. Lactic acid bacteria, for example, prefer acidic conditions, while yeast operates best in slightly acidic to neutral pH. Adjusting the pH can enable fermentation scientists to encourage or discourage certain kinds of microbial growth, giving them greater control over the community in the bioreactor and control what that product is good for and capable of. 

Temperature: The population growth rate of microbes and of microbial metabolism are highly temperature-dependent. (A microbe that evolved in the arctic is very likely to have different preferences than a microbe that evolved a boiling deep-sea vent!) And there are also subtler differences. For example, beer yeast ferments best around 18-22°C (64-72°F) for ales and 7-13°C (44 - 55°F) for lagers, while other industrial bacteria might prefer much higher or lower temperatures, depending on the species and end goal. The temperature also impacts the composition of various metabolites in the final product and hence affects the characteristics of that product. 

Agitation, aeration, and dissolved oxygen levels: Microbes take varying pleasure in oxygen. Some microbes hate oxygen (the “anaerobes”) meaning that they cannot survive in its presence. Some, like yeast, switch between aerobic and anaerobic metabolism. Oxygen also drives a microbial product's chemical profile: Aerobic conditions may be necessary for certain metabolites to be produced, while others require strict anaerobic conditions.

Nutrient supply: How much and which foods are available to microbes in the bioreactor will have immense and different impacts on fermentation outcomes. Carbon sources (e.g., glucose), nitrogen, minerals, and vitamins all influence microbial growth and product yield. And just as in humans, every microbe seems to have slightly different tastes. (Side note: There are plenty of “picky” microbes out there that are often referred to as “unculturable” because we have yet to discover what foods or conditions that they like exactly. Reminds me of a certain eight year-old that lives in my house…)

Sterility: Since, in monoculture, fermentation relies on a single species, contamination can ruin an entire batch. (This is true for co-culture as well, but contamination may be less likely because of increased numbers of species competing for space.) To keep contamination at bay, industrial bioreactors use some pretty hardcore sterilization techniques to prevent unwanted microbial competition.

Monoculture fermentation provides efficiency and predictability. However, if you look around at the world's ecologies, you’ve certainly noticed that nature rarely operates in single-species systems. As such, scientists are increasingly turning to co-culture fermentation to push the boundaries of biotechnology and bring the complexity and benefit of nature to bear in the bioreactor.

Co-culture fermentation: Complexity meets innovation

In co-culture fermentation, multiple microbial species work together, mimicking natural ecosystems to produce novel compounds, improve efficiency, or enhance sustainability. This approach is used in emerging applications like microbial consortia for biofuel production, plant probiotics, and even lab-grown meat. And of course, in what is still an unfortunately rare case, and nearest and dearest to our hearts here at Impello: co-cultured agricultural products.

However, the rarity of co-cultured products in this sector is not without reason. You probably noticed a recurring theme that monoculture fermentation and its techniques are all about control. It is not so much that the techniques for managing co-culture fermentation are drastically different, but more so that, because of all the different species involved, the complexity of co-culture is vastly increased and thus, “control” becomes a far more challenging project. So, you might ask: why bother? What makes us believe so strongly at Impello that this difficulty is worth taking on?

Why use co-culture fermentation?

Metabolic complementation: We talked a little bit above about the byproducts of microbes or their “metabolites”. Microbes produce an incredibly diverse assortment of these molecules that are bioactive in thousands of ways. To name just a few: these compounds fight disease, feed other organisms, break down organic tissues, and build and rebuild others. In a co-culture, sometimes certain species of microbes consume waste products from others, increasing overall efficiency of the system. In kombucha, for instance, a well-known and popular co-culture, yeast produces alcohol, which is then converted into organic acids by bacteria, which re-feeds the yeast, creating a self-sustaining system. The same pattern plays out in agricultural systems: For example, how, phosphate-solubilizing bacteria (like those found in certain inoculants), unlock bound-up forms of phosphorus that mycorrhizal fungi can then absorb and transport to plant hosts. In return, the plant root and fungal exudates carbon feed the bacteria—forming a self-reinforcing nutrient-sharing loop.

Enhanced stability: We talked briefly about how it can be very difficult to control contamination in monoculture fermentation. This parallels the same vulnerability we see in monocultural crops: if you plant only a single variety across many many acres— every single one of those acres has the same vulnerabilities to diseases and pests. If you plant many diverse types, a single pest or pathogen is unlikely to have the capacity to take over. A lack of microbial (and plant) diversity is also what often renders controlled environment agriculture so vulnerable to outbreaks. But in a multi-species microbial co-culture, the diversity of the collective means that no one species has a chance to gain a dominant foothold. Put another way, the community as a whole, and not any one so-called “pesticidal” microbe, has a chance to outcompete contaminants better than a monoculture. Apart from its species diversity, a diverse community also makes a more diverse array of metabolites that can have plant beneficial or disease suppressing abilities. It is kind of like the difference between, say, a pill for humans that has only vitamin C, versus a multivitamin that has, not only, vitamin C and its benefits, but also a myriad of other health-supporting compounds and their properties. While there is no doubt growing multiple microbes together is complex, on the whole, this complexity can make co-cultures more robust and less vulnerable as industrial applications and more useful in their end-use situations, i.e., agricultural fields. 

Expanded product diversity: Typically for a single species of microbe, the profile of what they can produce is relatively limited, and therefore the range of applications is narrow. In addition, some valuable, industrially produced compounds require interactions between multiple species to complete whole synthesis pathways. For example, some biofuels and pharmaceuticals can only be efficiently synthesized in co-culture systems because they need multiple types of microbes to complete the multiple metabolic steps needed to get to the final destination. Co-culture offers a way to produce a far wider array of useful molecules for agriculture that are not always possible with single species fermentation. 

Microbial co-culture challenges:

We’d be remiss if we didn’t talk about some of the complexities of co-culture and what makes them such a challenging project. For one, when you introduce multiple species into the same bioreactor, you must automatically confront the challenge of community balance. Keeping a community balanced means keeping multiple species at the appropriate ratios throughout the fermentation process and requires careful consideration, as slight changes in any conditions can cause one strain to outcompete the others. 

In addition, sometimes the benefit is also the challenge. Earlier I spoke about the benefits of intermicrobial communication– that is how microbes secrete signaling molecules to one another when they are raised together in a bioreactor. This can be of huge benefit in that many of these molecules have positive effects on plant growth, or be anti-pathogenic, but, because these are the molecules that microbes use to talk to one another it means these molecules also often influence microbial behavior, sometimes unpredictably. For some producers without strategic optimization and QC protocols in place, this can cause batch to batch variation that can frustrate the goal of making a consistent product. However, for companies willing to put in the time and effort to understand co-culture optimization techniques, it is possible to grow complex communities and keep the results dependable. And finally, scaling up can also be a huge challenge in co-cultures. Because bacterial behavior can be dependent on the population size, what works on the lab bench may not always function the same way in a 10,000-liter bioreactor. Often it takes an entirely new set of protocols and parameters to manage a larger batch size. Reproducing the right conditions at scale requires sophisticated modeling and monitoring.

Despite these challenges, co-culture fermentation holds immense promise. Scientists are engineering microbial consortia to perform tasks beyond the reach of monocultures, from breaking down complex plant materials into biofuels to creating entirely new food products. It is key to remember that in agricultural systems diversity = resilience and yield over time. There is no reason that we can’t bring these possibilities to fruition for farmers through microbial co-culture. 

The future of fermentation science

As researchers continue to refine fermentation processes, we may see even greater integration of synthetic biology, microbial ecology, artificial intelligence, and automation. Designed microbial communities “synthetic communities” or “SynComs” could transform many industries, not just agriculture, making fermentation not just a method of preserving food but a cornerstone of the future bioeconomy.

Whether in a block of aged cheese, a bottle of craft beer, or in a high-tech bioreactor rearing the best possible communities for agriculture, co-culture fermentation is a testament to the transformative power of microbes. Stay tuned for our next installment in which we will talk about some of the untapped possibilities for how microbes can be adapted to meet agricultural challenges.