A starter culture is a preparation of living microbes that is added to raw food material to accelerate, steer, and/or ensure consistent characteristics of the resulting fermentation product, which is often characterized by the timely production of desired microbial metabolites. Lactic acid bacteria are widely used as starter cultures in fermented dairy, sausage, vegetables, soy sauce, sourdough, and wine (Leroy and De Vuyst, 2004). For higher industrial productivity, a starter culture is typically optimized to perform certain conversions. While various traits, e.g., stress tolerance, adaptation to technological parameters, and key-metabolite production can be optimized, growth and acidification rate remain predominant selection criteria across products and organisms (Vinicius De Melo Pereira et al., 2020).

Growth rate is a fundamental property that reflects fitness and is highly optimized in unicellular organisms (Molenaar et al., 2009). In applications, the selection of starter cultures toward a faster growth rate under industrially relevant conditions ensures economical production. The corresponding faster acidification ensures that the starter culture outcompetes spoilage or pathogenic microorganisms through depletion of carbon source and pH inhibition. However, several disadvantages are seen with growth and acidification optimization. One common example is the loss of plasmid-encoded traits due to the cellular burden of plasmid maintenance (Bachmann et al., 2011; Ow et al., 2006). Additionally, the rise and subsequent invasion of cheater subpopulations that exploit public-good producing subpopulations may occur and lead to lower product formation and population collapse at worst (Bachmann et al., 2011, Bachmann et al., 2016; Smith and Schuster, 2019). In the case of dairy fermentations, a plasmid-encoded membrane-bound protease (PrtP) is a burden to the cell and often lost upon propagation, which leads to the emergence of faster growing prt− mutants that outcompete the slower growing prt+ population (Bachmann et al., 2011).

Adaptation to environmental conditions can lead to growth rate dependent switching in metabolic strategies, which is suggested to occur due to trade-offs in cellular economy and resource allocation to maximise fitness (Bachmann et al., 2016). In lactic acid bacteria (LAB), this manifests in overflow metabolism, particularly lactic acid fermentation. Pyruvate conversion to lactic acid is predominating during higher growth rates despite yielding 1 ATP less compared to its conversion to acetic acid. This may be counterintuitive since it seems a waste of energy. However, lactic acid fermentation requires fewer enzymes to be synthesized (Chen et al., 2021) and allows the maintenance of a high glycolytic flux, whereby it supports higher growth rates. The difference in enzymes required for the two strategies relates to constraints in the cytoplasmic proteome which is conceptualized as a “bacterial growth law” (Scott et al., 2010). This law describes that a high ribosome abundance (ribosomal proteins constitute a larger fraction of the cellular proteome) correlates with fast growth, which effectively decreases the proteome fraction that is allocated to metabolic proteins (Scott et al., 2010). The underlying reason is described to be constraint on protein content and cell size, which cannot increase indefinitely. This suggests that a high ribosomal fraction during fast growth of a dairy starter could come at the expense of starter functionality. These growth-law dependent trade-offs in proteome allocation have initially been demonstrated in E. coli, and were confirmed to also apply to Bacillus subtilis (Reuß et al., 2017), Aerobacter aerogenes, various yeasts (Elsemman et al., 2022; Xia et al., 2022), fungi, and Euglena gracilis (Scott et al., 2010). However, despite these consistent findings in these heterotrophic organisms, the same observation could not be replicated in Methanococcus maripaludis, which is a slow-growing chemolithoautotrophic archaeon that is highly adapted to energy-limiting environments (Müller et al., 2021). In Methanococcus maripaludis, ribosome numbers were largely invariant with growth rate, and protein synthesis rates were altered through modulation of ribosomal activity rather than ribosomal abundance. This suggests that adaptation to different lifestyles and habitats may coincide with the evolution of alternative resource allocation strategies. In the case of dairy LAB, cells evolved in lactose-abundant and nutrient rich environments, that are possibly different to feast-and-famine regimes experienced by many environmental microorganisms. For lactococci, “chemical warfare” is considered to be a competitive strategy which is facilitated by rapid acidification to create unsuitable environment for competitors (Goel et al., 2012). In lactococci, it has been reported that the proteome hardly changed with growth rate (Goel et al., 2015).

Given the broad use of lactic acid bacteria in the food industry and the ambiguity between growth rate and flavour formation, we here assess whether the described bacterial growth laws apply in lactococci. For this we determined flavour formation in a defined medium and a dairy environment and compared it to the corresponding proteomes of Lactococcus cremoris NCDO712 pre-cultured at different growth rates.