Combination with time-resolved microscopic inspection at using live/dead staining, enabled the generation of additional data related to the organisms’ physiological state, viability and population heterogeneity [25,30,31]. This set-up was employed to evaluate growth and viability during CO2 limitation under aerobic and anaerobic conditions. To this end, AnoporeTM slides on MRS-agar plates were inoculated with different dilutions of cells and incubated 18325633 in jars filled with gas-mixtures varying in CO2 and O2 content. At regular intervals the viability and size of the colonies were 
determined using a live/dead baclight stain as described in Materials Methods. The sum of the propidium iodide stained pixels and the SYTO9 stained pixels was used to estimate the size of the colony. The fraction of SYTO9 over all stained pixels was used as a relative measure of viability. CO2 supplementation to the gas phase (5 ) was found to stimulate growth under both aerobic (air) and anaerobic (N2) conditions. When plates were transferred to a CO2 depleted environment, growth stagnated after 7 hours, both in aerobic and anaerobic conditions. In the presence of supplemented CO2, microcolonies continued growth with an 58-49-1 estimated growth rate of 0.79 h21 in the anaerobic, and 0.74 h21 in the aerobic environment, which is comparable to growth rate in liquid culture (data not shown). This growth rate was estimated by fitting an exponential trend line through the average colony size (Figure 1, panels A and B). Growth stagnation was accompanied by loss of membrane integrity observed in microcolonies that are grown without CO2 supplementation, whereas microcolonies grown in CO2 supplemented environments sustained viability above 90 throughout the experiment (Figure 1, panels C and D). Notably, microcolonies grown in aerobic atmosphere displayed reduced loss of viability albeit with a higher degree of heterogeneity, as compared to microcolonies grown in a nitrogen atmosphere (Figure 1 C and D). This observation was remarkable since it has been documented that L. johnsonii produces hydrogen peroxide in the presence of oxygen [24], which was presumed to reduce growth rate and induce considerable cell death under aerobic conditions. Taken together, these results suggest that CO2 depletion leads to loss of membrane integrity and growth stagnation, while oxygenation appears to support extended viability as compared to anaerobic conditions.Oxygen Overcomes the Acetate Dependency of L. johnsonii NCCIn addition to CO2 dependency, growth of many lactobacilli also depends on the presence of acetate in the growth medium [14]. L. johnsonii was unable to grow in chemically defined medium without acetate supplementation. Notably, the addition of as little as 12 mM sodium acetate (1/1000 of the regular sodium acetate concentration in the chemically defined medium) allowed for recovery of growth, albeit at a slower rate and yielding lower final biomass concentrations. Acetate supplementation at a 100-fold lower level as compared to its regular concentration in CDM (120 mM) completely restored normal anaerobic growth (Figure 3). These results show that although there is a Peptide M strict acetaterequirement for growth, this requirement is already fulfilled with concentrations that are substantially below the levels that are normally added to typical Lactobacillus-laboratory media, such as MRS or CDM. To assess whether the acetate requirement of L. johnsonii NCC 533 depended on the growth cond.Combination with time-resolved microscopic inspection at using live/dead staining, enabled the generation of additional data related to the organisms’ physiological state, viability and population heterogeneity [25,30,31]. This set-up was employed to evaluate growth and viability during CO2 limitation under aerobic and anaerobic conditions. To this end, AnoporeTM slides on MRS-agar plates were inoculated with different dilutions of cells and incubated 18325633 in jars filled with gas-mixtures varying in CO2 and O2 content. At regular intervals the viability and size of the colonies were determined using a live/dead baclight stain as described in Materials Methods. The sum of the propidium iodide stained pixels and the SYTO9 stained pixels was used to estimate the size of the colony. The fraction of SYTO9 over all stained pixels was used as a relative measure of viability. CO2 supplementation to the gas phase (5 ) was found to stimulate growth under both aerobic (air) and anaerobic (N2) conditions. When plates were transferred to a CO2 depleted environment, growth stagnated after 7 hours, both in aerobic and anaerobic conditions. In the presence of supplemented CO2, microcolonies continued growth with an estimated growth rate of 0.79 h21 in the anaerobic, and 0.74 h21 in the aerobic environment, which is comparable to growth rate in liquid culture (data not shown). This growth rate was estimated by fitting an exponential trend line through the average colony size (Figure 1, panels A and B). Growth stagnation was accompanied by loss of membrane integrity observed in microcolonies that are grown without CO2 supplementation, whereas microcolonies grown in CO2 supplemented environments sustained viability above 90 throughout the experiment (Figure 1, panels C and D). Notably, microcolonies grown in aerobic atmosphere displayed reduced loss of viability albeit with a higher degree of heterogeneity, as compared to microcolonies grown in a nitrogen atmosphere (Figure 1 C and D). This observation was remarkable since it has been documented that L. johnsonii produces hydrogen peroxide in the presence of oxygen [24], which was presumed to reduce growth rate and induce considerable cell death under aerobic conditions. Taken together, these results suggest that CO2 depletion leads to loss of membrane integrity and growth stagnation, while oxygenation appears to support extended viability as compared to anaerobic conditions.Oxygen 
Overcomes the Acetate Dependency of L. johnsonii NCCIn addition to CO2 dependency, growth of many lactobacilli also depends on the presence of acetate in the growth medium [14]. L. johnsonii was unable to grow in chemically defined medium without acetate supplementation. Notably, the addition of as little as 12 mM sodium acetate (1/1000 of the regular sodium acetate concentration in the chemically defined medium) allowed for recovery of growth, albeit at a slower rate and yielding lower final biomass concentrations. Acetate supplementation at a 100-fold lower level as compared to its regular concentration in CDM (120 mM) completely restored normal anaerobic growth (Figure 3). These results show that although there is a strict acetaterequirement for growth, this requirement is already fulfilled with concentrations that are substantially below the levels that are normally added to typical Lactobacillus-laboratory media, such as MRS or CDM. To assess whether the acetate requirement of L. johnsonii NCC 533 depended on the growth cond.
