Bacterial proteome adaptation during fermentation in dairy environments.

The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.

Spatially structured microbial consortia and their role in food fermentations.

Microbial consortia are important for the fermentation of foods. They bring combined functionalities to the fermented product, but stability and product consistency of fermentations with complex consortia can be hard to control. Some of these consortia, such as water- and milk-kefir and kombucha, grow as multispecies aggregates or biofilms, in which micro-organisms taking part in a fermentation cascade are spatially organized. The spatial organization of micro-organisms in these aggregates can impact what metabolic interactions are realized in the consortia, ultimately affecting the growth dynamics and evolution of microbes. A better understanding of such spatially structured communities is of interest from the perspective of microbial ecology and biotechnology, as multispecies aggregates can be used to valorize energy-rich substrates, such as plant-based substrates or side streams from the food industry.

The hierarchy of sugar catabolization in Lactococcus cremoris.

IMPORTANCE: The availability of nutrients to microorganisms varies considerably between different environments, and changes can occur rapidly. As a general rule, a fast growth rate-typically growth on glucose-is associated with the repression of other carbohydrate utilization genes, but it is not clear to what extent catabolite repression is exerted by other sugars. We investigated the hierarchy of sugar utilization after substrate transitions in Lactococcus cremoris. For this, we determined the proteome and carbohydrate utilization capacity after growth on different sugars. The results show that the preparedness of cells for the utilization of "slower" sugars is not strictly determined by the growth rate. The data point to individual proteins relevant for various sugar transitions and suggest that the evolutionary history of the organism might be responsible for deviations from a strictly growth rate-related sugar catabolization hierarchy.

Manganese Modulates Metabolic Activity and Redox Homeostasis in Translationally Blocked Lactococcus cremoris, Impacting Metabolic Persistence, Cell Culturability, and Flavor Formation.

Manganese (Mn) is an essential trace element that is supplemented in microbial media with varying benefits across species and growth conditions. We found that growth of Lactococcus cremoris was unaffected by manganese omission from the growth medium. The main proteome adaptation to manganese omission involved increased manganese transporter production (up to 2,000-fold), while the remaining 10 significant proteome changes were between 1.4- and 4-fold. Further investigation in translationally blocked (TB), nongrowing cells showed that Mn supplementation (20 µM) led to approximately 1.5 X faster acidification compared with Mn-free conditions. However, this faster acidification stagnated within 24 h, likely due to draining of intracellular NADH that coincides with substantial loss of culturability. Conversely, without manganese, nongrowing cells persisted to acidify for weeks, albeit at a reduced rate, but maintaining redox balance and culturability. Strikingly, despite being unculturable, α-keto acid-derived aldehydes continued to accumulate in cells incubated in the presence of manganese, whereas without manganese cells predominantly formed the corresponding alcohols. This is most likely reflecting NADH availability for the alcohol dehydrogenase-catalyzed conversion. Overall, manganese influences the lactococcal acidification rate, and flavor formation capacity in a redox dependent manner. These are important industrial traits especially during cheese ripening, where cells are in a non-growing, often unculturable state. IMPORTANCE In nature as well as in various biotechnology applications, microorganisms are often in a nongrowing state and their metabolic persistence determines cell survival and functionality. Industrial examples are dairy fermentations where bacteria remain active during the ripening phases that can take up to months and even years. Here we investigated environmental factors that can influence lactococcal metabolic persistence throughout such prolonged periods. We found that in the absence of manganese, acidification of nongrowing cells remained active for weeks while in the presence of manganese it stopped within 1 day. The latter coincided with the accumulation of amino acid derived volatile metabolites. Based on metabolic conversions, proteome analysis, and a reporter assay, we demonstrated that the manganese elicited effects were NADH dependent. Overall the results show the effect of environmental modulation on prolonged cell-based catalysis, which is highly relevant to non-growing cells in nature and biotechnological applications.

Metabolic Conversions by Lactic Acid Bacteria during Plant Protein Fermentations.

To secure a sustainable food supply for the rapidly growing global population, great efforts towards a plant-based diet are underway. However, the use of plant proteins comes with several challenges, such as improvement or removal of undesired flavours, and generation of desired texture properties. Fermentation holds large potential to alter these properties, but compared to dairy fermentations, our knowledge on strain properties in different plant-based substrates is still limited. Here, we explored different lactic acid bacteria for their ability to grow, produce flavour compounds, or remove off-flavour compounds from different plant proteins. For this, 151 LAB strains from dairy and non-dairy origins were cultured in plant protein plus coconut oil emulsions supplemented with glucose. Pea, chickpea, mung, fava, and soybean proteins were used in the study and bacterial strains for screening included the genera Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc. Efficient, high throughput, screening on plant proteins was developed and strains were assessed for their ability to (i) acidify and decrease the pH; (ii) express key enzymes involved in the formation of amino acid derived flavours, which included PepN (aminopeptidase N), PepXP (X-prolyl dipeptidyl peptidase), EstA (esterase), BcAT (branched chain aminotransferase), CBL (cystathione beta lyase), and ArAT (aromatic aminotransferase); and (iii) improve the overall aroma profile by generating dairy/cheesy notes and decreasing off flavours. Suitable screening conditions were determined, and highlighted the importance that a sufficient heat treatment must be applied to samples containing plant proteins, prior to fermentation, as an outgrowth of spore forming Bacillus cereus was observed if the material was only pasteurised. Enzyme activities for strains measured in rich broth vs. a buffered protein solution showed little-to-no correlation, which illustrated the importance of screening conditions to obtain predictive enzyme measurements. Aroma formation analysis allowed to identify strains that were able to increase key aromas such as diacetyl, acetoin, 2- and 3-methyl butanol, and 2,3-pentanedione, as well as decrease the off-flavours hexanal, pentanal, and nonanal. Our findings illustrate the importance of strain specific differences in the assessed functionalities and how a methodical approach to screening LAB can be applied to select suitable microorganisms that show promise in fermentation of plant proteins when applied in non-dairy cheese applications.

Chronic Release of Tailless Phage Particles from Lactococcus lactis.

Lactococcus lactis strains residing in the microbial community of a complex dairy starter culture named "Ur" are hosts to prophages belonging to the family Siphoviridae. L. lactis strains (TIFN1 to TIFN7) showed detectable spontaneous phage production and release (109 to 1010 phage particles/ml) and up to 10-fold increases upon prophage induction, while in both cases we observed no obvious cell lysis typically described for the lytic life cycle of Siphoviridae phages. Intrigued by this phenomenon, we investigated the host-phage interaction using strain TIFN1 (harboring prophage proPhi1) as a representative. We confirmed that during the massive phage release, all bacterial cells remain viable. Further, by monitoring phage replication in vivo, using a green fluorescence protein reporter combined with flow cytometry, we demonstrated that the majority of the bacterial population (over 80%) is actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, as evidenced by electron microscopy and lipid staining combined with chemical lipid analysis. Based on the collective observations, we propose a model of phage-host interaction in L. lactis TIFN1 where the phage particles are engulfed in membranes upon release, thereby leaving the producing host intact. Moreover, we discuss possible mechanisms of chronic, or nonlytic, release of LAB Siphoviridae phages and its impact on the bacterial host. IMPORTANCE In complex microbial consortia such as fermentation starters, bacteriophages can alter the dynamics and diversity of microbial communities. Bacteriophages infecting Lactococcus lactis are mostly studied for their detrimental impact on industrial dairy fermentation processes. In this study, we describe a novel form of phage-bacterium interaction in an L. lactis strain isolated from a complex dairy starter culture: when the prophages harbored in the L. lactis genome are activated, the phage particles are engulfed in lipid membranes upon release, leaving the producing host intact. Findings from this study provide additional insights into the diverse manners of phage-bacterium interactions and coevolution, which are essential for understanding the population dynamics in complex microbial communities like fermentation starters.

Selection for Cell Yield Does Not Reduce Overflow Metabolism in Escherichia coli.

Overflow metabolism is ubiquitous in nature, and it is often considered inefficient because it leads to a relatively low biomass yield per consumed carbon. This metabolic strategy has been described as advantageous because it supports high growth rates during nutrient competition. Here, we experimentally evolved bacteria without nutrient competition by repeatedly growing and mixing millions of parallel batch cultures of Escherichia coli. Each culture originated from a water-in-oil emulsion droplet seeded with a single cell. Unexpectedly we found that overflow metabolism (acetate production) did not change. Instead, the numerical cell yield during the consumption of the accumulated acetate increased as a consequence of a reduction in cell size. Our experiments and a mathematical model show that fast growth and overflow metabolism, followed by the consumption of the overflow metabolite, can lead to a higher numerical cell yield and therefore a higher fitness compared with full respiration of the substrate. This provides an evolutionary scenario where overflow metabolism can be favorable even in the absence of nutrient competition.

High throughput screening of technological and biopreservation traits of a large set of wild lactic acid bacteria from Brazilian artisanal cheeses.

This study aimed to evaluate technological (acidification, proteolysis, lipolysis, resistance to low pH, NaCl, and bile salts) and biopreservation (antimicrobial activity against foodborne pathogens) features of 1002 LAB by high throughput screening (HTS) methods. The LAB was isolated from 11 types of Brazilian artisanal cheeses (BAC) marketed in the main 5 producing regions. Remarkable intra-species variability in acidification rates have been found, which was most pronounced between isolates from Mina's artisanal cheeses, Caipira and Coalho cheeses. Lacticaseibacillus paracasei and Levilactobacillus brevis showed the fastest acidification rate; however, all isolates showed slower acidification rates than a lactococcal control strain (4.3 × lower). When testing inhibitory effects, > 75% of LAB isolates could inhibit the growth of Staphylococcus aureus ATCC 19095 and Listeria monocytogenes ATCC 7644. Two of these isolates, identified as Lactiplantibacillus plantarum and Lentilactobacillus buchneri, the sterile and neutral supernatants alone, were sufficient to inhibit L. monocytogenes growth. Principal component analysis (PCA) allowed the identification of functional groups based on proteolytic and lipolytic activity, osmotic stress resistance, and inhibition of L. monocytogenes. The type of cheese the isolates were recovered from influenced properties such as anti-listerial compounds and lipolytic enzyme production. The use of HTS and multivariate statistics allowed insights into a diverse set of LAB technological and biopreservation properties. These findings allow a profound knowledge of the heterogeneity of a large set of isolates, which can be further used to design starter cultures with varied and combined properties, such as biopreservation and technological features. Besides that, HTS makes it possible to analyze a vast panel of LAB strains, reducing costs and time within laboratory analysis, while avoiding the loss of information once all LAB are tested at the same time (differently from the traditional labor-intensive approach, in which a few numbers of strains is tested per time).

A centrifugation-based clearing method allows high-throughput acidification and growth-rate measurements in milk.

The turbidity of milk prohibits the use of optical density measurements for strain characterizations. This often limits research to laboratory media. Here, we cleared milk through centrifugation to remove insoluble milk solids. This resulted in a clear liquid phase, termed milk serum, in which optical density measurements can be used to track microbial growth until a pH of 5.2 is reached. At pH 5.2 coagulation of the soluble protein occurs, making the medium opaque again. We found that behavior in milk serum was predictive of that in milk for 39 Lactococcus lactis (R2 = 0.81) and to a lesser extent for 42 Lactiplantibacillus plantarum (formerly Lactobacillus plantarum; R2 = 0.49) strains. Hence, milk serum can be used as an optically clear alternative to milk for comparison of microbial growth and metabolic characteristics. Characterization of the growth rate, specific acidification rate for optical density at a wavelength of 600 nm, and the amount of acid produced per unit of biomass for all these strains in milk serum, showed that almost all strains could grow in milk, with higher specific acidification and growth rates of Lc. lactis strains compared with Lb. plantarum strains. Nondairy Lc. lactis isolates had a lower growth and specific acidification rate than dairy isolates. The amount of acid produced per unit biomass was relatively high and similar for Lc. lactis dairy and nondairy isolates, as opposed to Lb. plantarum isolates. Lactococcus lactis ssp. lactis showed slightly lower growth and acidification rates when compared with ssp. cremoris. For Lc. lactis strains a doubling of the specific acidification rate occurred with a doubling of the maximum growth rate. This relation was not found for Lb. plantarum strains, where the acidification rate remained relatively constant across 39 strains with growth rates ranging from 0.2 h-1 to 0.3 h-1. We conclude that milk serum is a valuable alternative to milk for high-throughput strain characterization during milk fermentation.

Population dynamics of microbial cross-feeding are determined by co-localization probabilities and cooperation-independent cheater growth.

As natural selection acts on individual organisms the evolution of costly cooperation between microorganisms is an intriguing phenomenon. Introduction of spatial structure to privatize exchanged molecules can explain the evolution of cooperation. However, in many natural systems cells can also grow to low cell concentrations in the absence of these exchanged molecules, thus showing "cooperation-independent background growth". We here serially propagated a synthetic cross-feeding consortium of lactococci in the droplets of a water-in-oil emulsion, essentially mimicking group selection with varying founder population sizes. The results show that when the growth of cheaters completely depends on cooperators, cooperators outcompete cheaters. However, cheaters outcompete cooperators when they can independently grow to only ten percent of the consortium carrying capacity. This result is the consequence of a probabilistic effect, as low founder population sizes in droplets decrease the frequency of cooperator co-localization. Cooperator-enrichment can be recovered by increasing the founder population size in droplets to intermediate values. Together with mathematical modelling our results suggest that co-localization probabilities in a spatially structured environment leave a small window of opportunity for the evolution of cooperation between organisms that do not benefit from their cooperative trait when in isolation or form multispecies aggregates.

Proteome constraints reveal targets for improving microbial fitness in nutrient-rich environments.

Cells adapt to different conditions via gene expression that tunes metabolism for maximal fitness. Constraints on cellular proteome may limit such expression strategies and introduce trade-offs. Resource allocation under proteome constraints has explained regulatory strategies in bacteria. It is unclear, however, to what extent these constraints can predict evolutionary changes, especially for microorganisms that evolved under nutrient-rich conditions, i.e., multiple available nitrogen sources, such as Lactococcus lactis. Here, we present a proteome-constrained genome-scale metabolic model of L. lactis (pcLactis) to interpret growth on multiple nutrients. Through integration of proteomics and flux data, in glucose-limited chemostats, the model predicted glucose and arginine uptake as dominant constraints at low growth rates. Indeed, glucose and arginine catabolism were found upregulated in evolved mutants. At high growth rates, pcLactis correctly predicted the observed shutdown of arginine catabolism because limited proteome availability favored lactate for ATP production. Thus, our model-based analysis is able to identify and explain the proteome constraints that limit growth rate in nutrient-rich environments and thus form targets of fitness improvement.

Serial propagation in water-in-oil emulsions selects for Saccharomyces cerevisiae strains with a reduced cell size or an increased biomass yield on glucose.

In S. cerevisiae and many other micro-organisms an increase in metabolic efficiency (i.e. ATP yield on carbon) is accompanied by a decrease in growth rate. From a fundamental point of view, studying these yield-rate trade-offs provides insight in for example microbial evolution and cellular regulation. From a biotechnological point of view, increasing the ATP yield on carbon might increase the yield of anabolic products. We here aimed to select S. cerevisiae mutants with an increased biomass yield. Serial propagation of individual cells in water-in-oil emulsions previously enabled the selection of lactococci with increased biomass yields, and adapting this protocol for yeast allowed us to enrich an engineered Crabtree-negative S. cerevisiae strain with a high biomass yield on glucose. When we started the selection with an S. cerevisiae deletion collection, serial propagation in emulsion enriched hxk2Δ and reg1Δ strains with an increased biomass yield on glucose. Surprisingly, a tps1Δ strain was highly abundant in both emulsion- and suspension-propagated populations. In a separate experiment we propagated a chemically mutagenized S. cerevisiae population in emulsion, which resulted in mutants with a higher cell number yield on glucose, but no significantly changed biomass yield. Genome analyses indicate that genes involved in glucose repression and cell cycle processes play a role in the selected phenotypes. The repeated identification of mutations in genes involved in glucose-repression indicates that serial propagation in emulsion is a valuable tool to study metabolic efficiency in S. cerevisiae.

Stationary Lactococcus cremoris: Energetic State, Protein Synthesis Without Nitrogen and Their Effect on Survival.

During storage and ripening of fermented foods, Lactococcus cremoris is predominantly in a non-growing state. L. cremoris can become stationary due to starvation or acidification, and its metabolism in these non-growing states affects the fermented product. Available studies on the response of L. cremoris to acid and starvation stress are based on population level data. We here characterized the energetic state and the protein synthesis capacity of stationary L. cremoris cultures at the single cell level. We show that glucose starved stationary cells are energy-depleted, while acid-induced stationary cells are energized and can maintain a pH gradient over their membrane. In the absence of glucose and arginine, a small pH gradient can still be maintained. Subpopulations of stationary cells can synthesize protein without a nitrogen source, and the subpopulation size decreases with increasing stationary phase length. Protein synthesis capacity during starvation only benefits culturability after 6 days. These results highlight significant differences between glucose starved stationary and acid-induced stationary cells. Furthermore, they show that the physiology of stationary phase L. cremoris cells is multi-facetted and heterogeneous, and the presence of an energy source during stationary phase impacts the cells capacity to adapt to their environment.

Microbial competition reduces metabolic interaction distances to the low µm-range.

Metabolic interactions between cells affect microbial community compositions and hence their function in ecosystems. It is well-known that under competition for the exchanged metabolite, concentration gradients constrain the distances over which interactions can occur. However, interaction distances are typically quantified in two-dimensional systems or without accounting for competition or other metabolite-removal, conditions which may not very often match natural ecosystems. We here analyze the impact of cell-to-cell distance on unidirectional cross-feeding in a three-dimensional aqueous system with competition for the exchanged metabolite. Effective interaction distances were computed with a reaction-diffusion model and experimentally verified by growing a synthetic consortium of 1 µm-sized metabolite producer, receiver, and competitor cells in different spatial structures. We show that receivers cannot interact with producers located on average 15 µm away from them, as product concentration gradients flatten close to producer cells. We developed an aggregation protocol and varied the receiver cells' product affinity, to show that within producer-receiver aggregates even low-affinity receiver cells could interact with producers. These results show that competition or other metabolite-removal of a public good in a three-dimensional system reduces metabolic interaction distances to the low µm-range, highlighting the importance of concentration gradients as physical constraint for cellular interactions.

A Novel Method for Long-Term Analysis of Lactic Acid and Ammonium Production in Non-growing Lactococcus lactis Reveals Pre-culture and Strain Dependence.

In various (industrial) conditions, cells are in a non-growing but metabolically active state in which de novo protein synthesis capacity is limited. The production of a metabolite by such non-growing cells is dependent on the cellular condition and enzyme activities, such as the amount, stability, and degradation of the enzyme(s). For industrial fermentations in which the metabolites of interest are mainly formed after cells enter the stationary phase, the investigation of prolonged metabolite production is of great importance. However, current batch model systems do not allow prolonged measurements due to metabolite accumulation driving product-inhibition. Here we developed a protocol that allows high-throughput metabolic measurements to be followed in real-time over extended periods (weeks). As a validation model, sugar utilization and arginine consumption by a low density of translationally blocked Lactococcus lactis was designed in a defined medium. In this system L. lactis MG1363 was compared with its derivative HB60, a strain described to achieve higher metabolic yield through a shift toward heterofermentative metabolism. The results showed that in a non-growing state HB60 is able to utilize more arginine than MG1363, and for both strains the decay of the measured activities were dependent on pre-culture conditions. During the first 5 days of monitoring a ∼25-fold decrease in acidification rate was found for strain HB60 as compared to a ∼20-fold decrease for strain MG1363. Such measurements are relevant for the understanding of microbial metabolism and for optimizing applications in which cells are frequently exposed to long-term suboptimal conditions, such as microbial cell factories, fermentation ripening, and storage survival.

Lifestyle, metabolism and environmental adaptation in Lactococcus lactis.

Lactococcus lactis serves as a paradigm organism for the lactic acid bacteria (LAB). Extensive research into the molecular biology, metabolism and physiology of several model strains of this species has been fundamental for our understanding of the LAB. Genomic studies have provided new insights into the species L. lactis, including the resolution of the genetic basis of its subspecies division, as well as the control mechanisms involved in the fine-tuning of growth rate and energy metabolism. In addition, it has enabled novel approaches to study lactococcal lifestyle adaptations to the dairy application environment, including its adjustment to near-zero growth rates that are particularly relevant in the context of cheese ripening. This review highlights various insights in these areas and exemplifies the strength of combining experimental evolution with functional genomics and bacterial physiology research to expand our fundamental understanding of the L. lactis lifestyle under different environmental conditions.

Enhancement of amino acid production and secretion by Lactococcus lactis using a droplet-based biosensing and selection system.

Amino acids are attractive metabolites for the pharmaceutical and food industry field. On one hand, the construction of microbial cell factories for large-scale production aims to satisfy the demand for amino acids as bulk biochemical. On the other hand, amino acids enhance flavor formation in fermented foods. Concerning the latter, flavor formation in dairy products, such as cheese is associated with the presence of lactic acid bacteria (LAB). In particular, Lactococcus lactis, one of the most important LAB, is used as a starter culture in fermented foods. The proteolytic activity of some L. lactis strains results in peptides and amino acids, which are flavor compounds or flavor precursors. However, it is still a challenge to isolate bacterial cells with enhanced amino acid production and secretion activity. In this work, we developed a growth-based sensor strain to detect the essential amino acids isoleucine, leucine, valine, histidine and methionine. Amino acids are metabolites that can be secreted by some bacteria. Therefore, our biosensor allowed us to identify wild-type L. lactis strains that naturally secrete amino acids, by using co-cultures of the biosensor strain with potential amino acid producing strains. Subsequently, we used this biosensor in combination with a droplet-based screening approach, and isolated three mutated L. lactis IPLA838 strains with 5-10 fold increased amino acid-secretion compared to the wild type. Genome re-sequencing revealed mutations in genes encoding proteins that participate in peptide uptake and peptide degradation. We argue that an unbalance in the regulation of amino acid levels as a result of these gene mutations may drive the accumulation and secretion of these amino acids. This biosensing system tackles the problem of selection for overproduction of secreted molecules, which requires the coupling of the product to the producing cell in the droplets.

Microdroplet screening and selection for improved microbial production of extracellular compounds.

Microorganisms produce extracellular compounds that affect the final product quality in fermentation processes. Selection of overproducing mutants requires coupling of the extracellular product to the producer genotype, which can be achieved by single-cell compartmentalization. Emulsions contain up to billions of microdroplets/mL which significantly increases the screening throughput compared to microtiter-plate-based selections. Factors affecting the success of screening in microdroplets include the nature of the producing organism (robust, no invasive growth), the product (not soluble in oil) and the product assay (preferably fluorescence based). Together these factors determine the required microdroplet production technique and sorting set-up. Because microdroplets allow relatively inexpensive ultrahigh-throughput screening, they are likely to become a standard tool in the strain selection toolbox of the fermentation industry.

Transcriptional response of Lactococcus lactis during bacterial emulsification.

Microbial surface properties are important for interactions with the environment in which cells reside. Surface properties of lactic acid bacteria significantly vary and some strains can form strong emulsions when mixed with a hydrocarbon. Lactococcus lactis NCDO712 forms oil-in-water emulsions upon mixing of a cell suspension with petroleum. In the emulsion the bacteria locate at the oil-water interphase which is consistent with Pickering stabilization. Cells of strain NCDO712 mixed with sunflower seed oil did not stabilize the oil droplets. This study shows that the addition of either ethanol or ammonium sulfate led to cell aggregation, which subsequently allowed stabilizing oil-in-water emulsions. From this, we conclude that bacterial cell aggregation is important for emulsion droplet stabilization. To determine how bacterial emulsification influences the microbial transcriptome RNAseq analysis was performed on lactococci taken from the oil-water interphase. In comparison to cells in suspension 72 genes were significantly differentially expressed with a more than 4-fold difference. The majority of these genes encode proteins involved in transport processes and the metabolism of amino acids, carbohydrates and ions. Especially the proportion of genes belonging to the CodY regulon was high. Our results also point out that in a complex environment such as food fermentations a heterogeneous response of microbes might be caused by microbe-matrix interactions. In addition, microdroplet technologies are increasingly used in research. The understanding of interactions between bacterial cells and oil-water interphases is of importance for conducting and interpreting such experiments.

Ampicillin-treated Lactococcus lactis MG1363 populations contain persisters as well as viable but non-culturable cells.

Lactococcus lactis is used as cell-factory and strain selections are regularly performed to improve production processes. When selection regimes only allow desired phenotypes to survive, for instance by using antibiotics to select for cells that do not grow in a specific condition, the presence of more resistant subpopulations with a wildtype genotype severely slows down the procedure. While the food grade organism L. lactis is not often exposed to antibiotics we characterized its response to ampicillin in more detail, to better understand emerging population heterogeneity and how this might affect strain selection procedures. Using growth-dependent viability assays we identified persister subpopulations in stationary and exponential phase. Growth-independent viability assays revealed a 100 times larger subpopulation that did not grow on plates or in liquid medium, but had an intact membrane and could maintain a pH gradient. Over one third of these cells restored their intracellular pH when we induced a temporary collapse, indicating that this subpopulation was metabolically active and in a viable but non-culturable state. Exposure of L. lactis MG1363 to ampicillin therefore results in a heterogeneous population response with different dormancy states. These dormant cells should be considered in survival-based strain selection procedures.