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Spus et al. Microbiome Res Rep 2023;2:33 https://dx.doi.org/10.20517/mrr.2023.20 Page 13 of 15

It was noted in other ecosystems that the decrease in diversity reduces the optimal utilization of resources
[32]
present . The absence of bacteriophage predation pressure in the blends led to domination by strains of
one particular genetic lineage (the fittest strains) without a complete clonal sweep of other lineages
[Figure 6]. Possibly these low abundant strains, as specialists with, e.g., citrate degradation ability, remained
in the blend by exploiting different (micro)niches. In such cases, a culture dominated by a single lineage not
necessarily has lower ecosystem efficiency but, due to division of labor, exploits the resources optimally. It
would be of interest to investigate the parameters of the blend efficiency (acidification, aroma formation)
and to define the effect of reduction in genetic lineage diversity on the blend’s functionality.

In our propagation experiment, we observed two contrasting stable states. In the case of the absence of
bacteriophage predation, the strains belonging to L. cremoris lineage 1 & 5 dominated the blend throughout
the entire time span of the propagation. Without bacteriophage predation pressure, the strain abundance of
“the winner” was not kept in control. On the other hand, the presence of phage predation led to a
maintenance of diversity at the level of genetic lineages by preventing domination of the otherwise fittest
strains.

In conclusion, phage predation, among other factors impacting complex microbial communities, can trigger
catastrophic shifts in the community population dynamics. Nevertheless, as shown with our extremely
detailed genetic lineage level culture composition analysis, recovery to an alternative stable state
(demonstrating PS dynamics) can take place even after a catastrophic shift. Furthermore, it is demonstrated
experimentally that bacteriophage predation on a complex microbial community can lead to prolonged
stabilization of culture diversity and thus functionality as indicated by a sustained relatively high Shannon
diversity index in one of the phage-challenged mixed cultures. Empirical data on the impact of
bacteriophage predation on multi-strain community diversity presented in this study add a further level of
detail to the PS vs. CD dynamics models describing diversity in microbial communities and stress the role
[9]
of microbe-microbe interactions, bacteriophage predation and (micro)niche adaptation in preventing clonal
sweeps.

DECLARATIONS
Acknowledgments
Parts of this work appeared as a chapter in the PhD thesis of Dr. M. Spus, entitled “Mixed culture
engineering for steering starter functionality” (ISBN 978-94-6257-833-3). This thesis was defended on 2-9-
2016 and it can be downloaded via this link: http://dx.doi.org/10.18174/383695.

Authors’ contributions
Wrote the manuscript and was responsible for the experimental design, performed the lab work (QPCR):
Spus M
Performed the lab work (preparation and propagation of the blends): Wardhana YR
Performed the lab work (plaque assays): Wolkers-Rooijackers JCM
Proofread the manuscript and contributed to the discussion: Abee T
Responsible for the manuscript outline, final proofreading and contribution to the interpretation and
discussion of the results: Smid EJ

Availability of data and materials
Raw data were stored in the TIFN database (https://www.tifn.nl), which is accessible upon request.