Lactic acid bacteria (LAB) are fermentative organisms which have been generally regarded as anaerobic bacteria, but most of them can grow under aerobic conditions (Sakamoto and Komagata, 1996). Beer is a relatively hostile medium for most microorganisms. The low pH will prevent most Gram-negative bacteria from growing, and the addition of hops will usually prevent LAB from spoiling the beer (Vaughan et al., 2005; Suzuki et al., 2006; Menz et al., 2010; Suzuki, 2011). However, some LAB possess a level of hop resistance, and therefore also possess the ability to spoil beer. 60–90% of bacteria isolated from spoiled beer are LAB (Suzuki, 2011). Among those LAB, Lactobacillus brevis is the most common bacteria and frequently detected in breweries (Hollerová and Kubizniaková, 2001; Suzuki et al., 2008a; Menz et al., 2010).

Recently, it has been suggested that hop resistance in LAB is inversely correlated to resistance toward oxidative compounds (Behr and Vogel, 2010). Consequently, it would be interesting to investigate the response of beer spoilage isolates toward oxidative compounds, as some of these are utilized as sanitizers in the food industry (Rossoni and Gaylarde, 2000; Kitis, 2004).

Another potential challenge is the relatively slow growth of the beer spoilage organisms. This means that often they are not detected readily in various culture media (Suzuki et al., 2008b). One way of facilitating the detection would be to use detection of growth of individual cells into micro-colonies, as the formations of macrocolonies require a longer incubation period in traditional CFU method. Recently, some studies have developed bioimaging methods for detecting the growth of individual cells in/on a solid matrix. Elfwing et al. (2004) designed a flow chamber microscopic method to observe growth and proliferation of single cells of Escherichia coli and Listeria innocua. Niven et al. (2006) developed a phase-contrast microscopy method to determine the first division time and individual lag times on agar media. Mertens et al. (2012) studied the colony growth dynamics based on optical density measurements on solid medium in microtiter plates. Koutsoumanis and Lianou (2013) used time-lapse microscopy videos to count the cells and to observe the division of Salmonella single cells directly on agar media. Ryssel et al. (2013) developed a microscopy method to monitor growth and death of individual Lactococcus lactis cells based on staining with propidium iodide (PI) in the agar media. In addition, another advantage of investigating individual cells growing on a solid substrate is the ability to analyze the heterogeneity of a given population, as each individual cell gives rise to a unique microcolony.

The current study therefore investigates the impact of oxidizing substances on the survival of beer spoilage LAB. The study describes an automated image-acquisition microscopic method that enables the analysis of growth as well as the death of individual cells while growing on the surface of a semisolid substrate.

As previously mentioned, there may be an inverse correlation between hop resistance and oxidation resistance. However, in this study a plasmid cured strain lacking the hop resistance gene horA had a slightly higher sensitivity toward oxidizing compounds compared to the wild type JK09 (Figure 1). This does not support the idea of an inverse correlation, but the plasmid cured strain also exhibited similar hop resistance to the wild type (results not shown), which suggests that hor A is neither important for hop resistance nor oxidation resistance.

It was previously found that Lactobacillus brevis ATCC 14869 exhibit Smooth(S)-type colonies when grown under anaerobic conditions, whereas the majority of colonies exhibit a Rough(R)-type morphology under aerobic conditions (Jakava-Viljanen et al., 2002). In our study, the two Lactobacillus brevis strains HF01 and JK09 had distinctly different morphologies as JK09 exhibited S-type morphology and HF01 exhibited R-type morphology (Figure 2). However, their morphologies appeared to be less variable, as they retained the same morphology after PAA treatment, and the morphology was the same after aerobic and anaerobic cultivation. The study of Jakava-Viljanen et al. (2002) indicated that an oxidative environment promotes the formation of R-type colonies, which could suggest that strains with R-type morphology has an increased survival after exposure to oxidative compounds. This is consistent with our findings, where HF01 (R-phenotype) was more tolerant toward PAA.

In addition, our microscopic results suggest that already when microcolonies are formed, a distinct difference in colony morphology can be observed, which may predict the resulting morphology of macrocolonies (Figures 2A,B and 3).

It is clear that the microscopic method is more rapid than the CFU method for detection of dividing cells, (Asano et al., 2009). The traditional approach required at least 2 days before visible colonies (consisting of millions of cells) could be detected. In the present study, small colonies continued to appear until five days after the PAA treatment. The origin of these colonies is cells that can be considered ‘hard-to-culture’ (Suzuki, 2011), but our microscopic results suggests that this phenomenon can be attributed to the large variation in lag time of individual cells, after PAA treatment. This could be the reason why some survival rates using the CFU method were 20% lower than that using the microscopic method and with similar big differences between repetitions in the CFU method (Table 2). It is possible that some individual cells started division so late that the (macro)colonies were too small to be observed on the last day of the experiment. However, the microscopic method could observe cell elongation and division down to few hours after the beginning of the experiment. The microscopic method also has the potential to provide more details of the growth of individual cells into microcolonies. In our study, we can clearly see the growth dynamics of individual cells (Figure 3), where cells divided and eventually formed a microcolony. We also observed in our experiments that a few cells elongated, but then stopped dividing (Figure 6). This type of subpopulation cannot be observed with a CFU method, although the cells may possess some amount of biochemical activity. This might be another reason why the survival rates using the CFU method in some cases were 20% lower than the microscopic method.

Several studies have shown that individual cells exhibit heterogeneity in how they deal with stress in the same environment (Lianou et al., 2006; Métris et al., 2008; Muñoz-Cuevas et al., 2013). We observed that there are relatively small differences in the growth curves and lag time of both strains in the control experiments (Figure 5), but extensive variation in growth behavior, survival, lag time and maximum growth rate was observed between the two strains after treatment with PAA (Table 2, Figure 5). This biological variability may be due to the genetic diversity between the strains, but the large variations in the resulting λ and μ_max of HF01 and JK09, especially after PAA treatments suggest that there is a phenotypic diversity that cannot be fully explained by presence of genes, as all of the individual cells of a strain can be considered clonal. After exposure to PAA, the lag time of the dividing cells of both strains increased pronouncedly. Interestingly, after the prolonged lag time, some cells of both strains exhibited the same μ_max as in the control experiments, whereas other cells grow at a much slower rate. This is interesting, because it suggests that even after repair mechanisms have enabled the individual cells to divide, the resulting daughter cells in a microcolony continue to divide at a rate that is predicted by the initial divisions, even though PAA has been removed. This result is inconsistent with the results from Kutalik et al. (2005), which points out that the extent of the lag phase only influence the first cell cycle and the subsequent division is uncorrelated to the cell history. In addition, we found that the lag time was significantly shorter and the μmax was distinctly higher after the aerobic cultivation for both strains (Figure 5). This result indicates that aerobic cultivation makes Lactobacillus brevis more tolerant to PAA. It may be because the bacteria have already built up some kind of defense mechanism or repair mechanism in order to protect them against oxygen during the aerobic cultivation. For example, high NADH oxidase activity and NADH peroxidase activity were found in Lactobacillus brevis after aerobic cultivation (Sakamoto and Komagata, 1996; Hummel and Riebel, 2003). In addition, there are marked differences in λ and μ_max of neighboring microcolonies (Figure 6). The differences are not caused by the proximity between the resulting microcolonies, because even after 12 h and several divisions, the resulting microcolonies still have similar distances to the neighboring colonies. It is likely that there would be an interaction between adjacent colonies when they are so close that they must share nutrients (propinquity effect), but it is unlikely that this is the case within the timeframe of our experiments.

The microscopic method used in this study can therefore provide quantitative data suitable for analysis of growth of individual cells. It should be noted that we only quantified the amount of pixels that are covered by microcolonies in two dimensions. When the microcolonies become very big (several hundred cells), the microcolony started to take on the traditional three-dimensional structure (Figures 4A,C), where a simple measure of area is no longer adequate. However, during many divisions, the cells are primarily growing on the surface of the agar, as our cells are enclosed between the coverslip that constitutes the bottom of the well and the agar. Additionally, if the microcolonies expand in less coordinated fashion such as HF01, where there can be visible holes inside the microcolony, it is important to subtract the area of the holes to obtain a valid estimate of cell growth.

Propidium iodide is a popular red-fluorescent DNA counterstain for estimating the amount of dead cells in a bacterial population (Bunthof et al., 2001; Rault et al., 2007). Due to its molecular weight and charge, it only penetrates cells with a damaged plasma membrane. As PI does not fluoresce without the presence of DNA, we incorporated PI into our growth matrix, in order to determine when and how, individual cells begin to die. Surprisingly, the large proportion of cells that failed to divide after treatment with PAA, did not exhibit red fluorescence (Figures 4B,D). In JK09, the survival after anaerobic cultivation after PAA treatment was around 1%, so we would expect to observe 99% red cells. This observation suggests that PAA does not compromise the membrane integrity of cells that fail to divide, and PI would therefore be a poor indicator of the efficacy of PAA. To rule out potential artifacts in our experiments, we determined that PAA treated cells that were afterward exposed to 70% EtOH (which destroys the membrane) all stained red in our setup (results not shown). As microcolonies started to grow, we observed that a few individual cells turned red, which indicates that these cells have lost their membrane integrity (Figure 4). The cells appeared randomly within the microcolony, which does not suggest that they died from nutrition depletion or accumulation of toxic substances. If the cells should die from nutrient depletion or accumulation of toxic substances, it would be expected that the majority of the dead cells would be located in the center of the microcolony, this observation was previously reported by Ryssel et al. (2013).

In conclusion, the investigated beer spoilage LAB after different cultivations exhibit different sensitivity toward PAA, but there is no indication that the tolerance toward PAA is inversely correlated to the potential to spoil beer. On the other hand, the present study demonstrates a novel approach to investigate the formation of microcolonies as an indicator of physiological fitness. The method provides results faster than CFU determination, but seems to correlate very well with CFU. Furthermore, the method provides a tool to investigate the phenotypic heterogeneity of a clonal population, which can be expanded to many interesting aspects.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.