On the grain, competition between species reigns supreme. Many species appear to be capable of inhibiting L. kefiranofaciens, which keeps its abundance in check. Meanwhile, Lactococcus lactis, another member of the core community, produces toxins known as bacteriocins that target and strongly inhibit the growth of other members of the community.
Although the community on the grains remains stable throughout the fermentation process, the surrounding milk sees increases and declines in different members as the microbes move from a solid to a liquid environment. The researchers described the kefir grains as a "basecamp" from which the different microbes "colonize the milk in an orderly fashion." This migration happens in a set order of species, which appears to have a lot to do with the preferred diet of each group.
Next stop: Cooperation Station
Unlike the grain environment, the complex nutritional landscape of the milk pushes the microbes to cooperate rather than compete. First to colonize the milk is L. kefiranofaciens, which, as on the grain, is the most abundant species throughout the fermentation process. Surprisingly, despite its dominance, L. kefiranofaciens can't grow in milk on its own. Its growth has been suggested to be supported by rare species, many of which can grow in milk alone. These species break down proteins and sugars in the milk to produce smaller molecules, generally referred to as metabolites, that L. kefiranofaciens could use to grow.
The analysis of these metabolites, using several techniques collectively known as metabolomics, was crucial for resolving how and why all these microbes cooperate. By measuring the concentrations of key metabolites throughout the fermentation process, it became apparent that some of their patterns match the growth patterns of key kefir species. For example, the concentration of a key intermediate molecule that cells use to produce energy, called citrate, drops suddenly in the early stages of fermentation. This drop correlates with the growth of two kefir species, L. lactis, and Leuconostoc mesenteroides, which suggests that they could be using citrate to grow.
In the milk, cooperative interactions between species are far more common than competitive ones, indicating that there are benefits to be gleaned from the presence of others. For example, L. kefiranofaciens and L. mesenteroides share a mutually beneficial relationship in which the presence of one promotes the growth of the other. This partnership seems to be centered on cross-feeding between the two. L. kefiranofaciens breaks up proteins into amino acids that can be accessed by L. mesenteroides. In return, L. mesenteroides, now able to carry out fermentation, produces lactate which L. kefiranofaciens can consume.
The acid test
The production and accumulation of major fermentation products, including lactate and acetate, acidifies the milk and gives kefir its distinct, slightly sour taste. However, these products also play a role in regulating the growth of microbes in the process. They inhibit the growth of many species that are important in the early- to mid-stages of fermentation, including L. lactis, L. mesenteroides, and several rare species.
Meanwhile, other species appear to benefit from the accumulation of lactate and acetate. Metabolomics measurements reveal that different species of yeasts and Acetobacter bacteria begin to grow during a favorable window of lactate and acetate concentrations in the milk, indicating accumulation of these metabolites clear the way for late-growing species. Although, high concentrations of lactate and acetate would be unfavorable for everyone.
Like many of the other core species, Acetobacter fabarum cannot grow in milk on its own. Here, another beneficial cross-feeding interaction appears to be at play, with L. lactis, an earlier-growing species, providing lactate and specific amino acids for A. fabarum to consume. However, whether A. fabarum provides anything in return is not clear. In contrast to the mutualism between L. kefiranofaciens and L. mesenteroides, this interaction could be described as commensalism: one partner benefits, and the other is seemingly unaffected.
Studying interactions between microbes presents a near-limitless well of discovery, precisely because these interactions are everywhere and occur in many flavors: from cooperation to vicious competition, and everything in between. Humans also have the potential to leverage microbial communities for our benefit: for example, understanding how microbial communities associated with crop plants function could help us devise ways to increase hardiness and yields under environmental stress. One longer-term goal is to develop a comprehensive-enough understanding that we can build communities to perform specific tasks, whether that be degradation of pollutants in the environment, producing new chemical compounds, or improving our health.