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===IntroductionInitial Study===While most beer styles are fermented using one culture of ''Saccharomyces cerevisiae'' or ''S. pastorianus'', Flanders Red Ales are fermented with a [[Mixed Fermentation|mixed culture fermentation]]. At one brewery (presumed to be Rodenbach) studied by Martens et al., two beers were produced using mixed fermentation and blended together. The first "light beer" was 11°P and was less acidic, while the second "heavy beer" was 13°P and served as an Old Ale if unblended. Both beers were inoculated with an acid washed yeast slurry that was harvested from a previous fermentation of the "light beer", and contained about 5% lactic acid bacteria. The fermentation of these beers followed three stages: 1. a seven day ethanol fermentation that is dominated by ''Saccharomyces'', 2. a four to five week lactic acid fermentation that was dominated by ''Lactobacilli'', and 3. a twenty to twenty-four month fermentation that was dominated by ''Brettanomyces'', ''Lactobacilli'', ''Pediococcus'', and acetic acid bacteria. The development of the ''Brettanomyces'' and ''Pediococcus'' stage was similar to the development of these microbes in [[Lambic]] fermentation. The "light beer" was never allowed to go through the third phase of fermentation, and was instead chilled to 0°C and then used to blend with previous batches of the "heavy beer" <ref name="Martens">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1997.tb00939.x/abstract MICROBIOLOGICAL ASPECTS OF A MIXED YEAST—BACTERIAL FERMENTATION IN THE PRODUCTION OF A SPECIAL BELGIAN ACIDIC ALE. H. Martens, D. Iserentant andH. Verachtert. 1997.]</ref>. Interestingly, and perhaps frustratingly, Flanders red and brown ales have been the subject of had far fewer published studies far less than Belgian lambic beers, so the . The following information is based off of the Martens et al. study from 1997 (see referencereferences).

In the brewery studied by Martens et al., the "light beer" was inoculated with a harvested yeast slurry of multiple strains of ''S. cerevisiae'' at a rate of 8x10⁶ CFU/mL, and the "heavy beer" was inoculated with 1x10⁷ CFU/mL. Small numbers of ''Candida guillermondii'' and ''Candida datilla'' were reportedly in the yeast slurry, but their identification was questioned in the study and they were not found during primary fermentation. One interesting finding was that the ''S. cerevisiae'' strains used at this brewery formed sexual spores ([https://en.wikipedia.org/wiki/Ascospore ascospore]), which is quite unusual for brewing yeasts. The yeast in the "heavy beer" grew slower (3 days) and reached an overall cell count that was lower than the "light beer", which reached maximum cell count in 2 days. Yeast slurries with more lactic acid bacteria are generally used to inoculate the "heavy beer", and this may retard the yeast growth in the "heavy beer". The harvested slurry is always taken from the "light beer", which may be less adapted to the fermentation of the "heavy beer". Also, after 1 week the yeast flocculated and settled out better in the "light beer" than they did in the "heavy beer". Although lactic acid bacteria were in the yeast slurry, their growth started only started to appear after 4 days into the primary fermentation. No enterobacteria or acetic acid bacteria were found during this first phase of fermentation <ref name="Martens"></ref>.

The lactic acid bacteria found in the yeast slurry were consisted of 18 strains of ''L. delbruekii ssp delbruekii'' and 12 strains of ''L. delbruekii ssp bulgaricus''. Small amounts of ''Pediococcus'' were also identified in the slurry, but were impossible to isolate with enrichment until they were found in the primary fermentation. During primary fermentation, one strain of ''L. platnarumplantarum'', two strains of ''L. brevis'', and one strain of ''Pediococcus parvulus'' were identified <ref name="Martens"></ref>.

After the 7 days of the primary fermentation, the beer was transferred to a secondary fermenter and remained there for four to five weeks. Both the yeast and bacteria populations saw a decline during the transfer, and then a small and gradual growth in secondary with the final yeast count being 4.3x10⁵ in the "heavy beer" and 6.4x10³ in the "light beer". Lactic acid bacteria grew much faster and became dominate in the "light beer", whereas in the "heavy beer" they grew more slowly and yeast remained the dominate microbe. This is was explained by there being more sugars in the "heavy beer", which gave the yeast the advantage. Acetic acid bacteria were still not detected during secondary fermentation <ref name="Martens"></ref>. Lactic acid begins began to be produced as well during secondary fermentation, with about a third of it being L-lactic acid and two thirds of it being D-lactic acid <ref name="Verachtert">[http://www.lambic.info/images/7/78/Verachtert2014BelgianAcidBeersDailyReminiscencesofthePast.pdf BELGIAN ACIDIC BEERS Daily Reminiscences of the Past. Hubert Verachtert, Guy Derdelinckx. 2014.]</ref>.

The same study by Martens et al. looked at two casks during the third fermentation, where the . The "heavy beer" beer was transferred from the secondary fermentation vessel to the casks to age for two years. At the beginning of the third phase of fermentation, ''Saccharomyces'' cell counts began to drop while the appearance of ''Brettanomyces'' began. After 10 weeks in the casks, ''Lactobacilli'' greatly decreased, giving rise to strains of ''Pediococcus parvulus''. After 12 weeks for Cask A and 18 weeks for Cask B, the beer no longer contained ''Saccharomyces'', and ''Brettanomyces'' dominated. Specifically, ''B. lambicus'' (now classified as a strain of ''B. bruxellensis'' <ref name="snauwaert"></ref>) and ''B. bruxellensis'' were the dominate species, but much smaller counts of ''B. intermedius'' (now classified as ''B. anomala'') and ''B. custersianus'' were also found. The ''Brettanomyces'' species continued to be the primary microorganisms for 36 weeks in Cask A and 50 weeks in Cask B. After this time period, ''P. parvulus'' began to dominate. Acetic acid bacteria also began to make an appearance in the casks, being detected at 27 weeks in Cask A and 40 weeks in Cask B. The exact numbers of the acetic acid bacteria were not reported by this study since they mostly grow on the surface of the beer inside the cask, and possibly on the walls of the cask where diffused oxygen could reach them more easily, and samples were not taken from these sections of the casks. The difference in the time periods for the microbial populations was determined to be affected by the casks themselves, which differed in age, size, and possibly different microbe colonization inside them before they were filled <ref name="Martens"></ref>. During the third fermentation phase, lactic acid greatly increases increased from ~600 ppm to ~4500 ppm after 35 weeks, and then continued to slowly increases increase to ~5200 ppm at 60 weeks, at which time L-lactic is was only slightly less (~48%) than D-lactic acid <ref name="Verachtert"></ref>. Acetic acid levels reached 1600 ppm by the end of the third phase. The final pH of the "heavy beer" is was around 3.2-3.5 <ref name="snauwaert"></ref>.

[[Lambic ]] is a similar beer produced in Belgium, but is fermented using [[Spontaneous Fermentation|spontaneous fermentation]]. Enterbacteria Enterobacteria were not found in the Flanders red brewery that Martens et al. studied since spontaneous fermentation was not used. However, after the enterobacteria and primary ''Saccharomyces'' fermentation phases of lambic brewing are complete, the microbial populations of lambic and Flanders red/brown beers are similar during their aging processes. Both beers display a dominance by ''Brettanomyces'' and ''Pediococcus'' during the aging phases. Flanders Red Ales differ by having a large portion of the acid production performed by ''Lactobacilli'', where as in lambic the acid production is performed by ''Pediodoccus damnosus''. Flanders Red Ales red ales are also characterized by having ''P. parvulus'' instead of ''P. damnosus'' (although this may have been misidentified; see [[Flanders_Red_Ale#Modern_Analysis|Modern Analysis ]] below), however Martens et al. noted that the two species have no clear difference as far as their effects on fermentation go. Martens also noted that ''Brettanomyces'' began to disappear from old English Porter when the beer moved from wooden casks to metallic ones. It is thought then that the wooden casks are vital to ''Brettanomyces'' in Flanders Red Ale brewing, perhaps due to the presence of ''Pediococci'', with which ''Brettanomyces'' "cooperates" to ferment dextrins in the beer during the aging phase <ref name="Martens"></ref>. Belgian brewers have even married the Flanders red ale and lambic by blending the two beers together. The Flanders "acid beer" is fermented with cherries, and later on lambic is added and the blend is allowed to referment in the bottle, creating something truly special <ref name="Verachtert"></ref>.

Belgian brewers have even married the Flanders Red Ale and Lambic ===Modern Analysis===A more recent study by blending the two beers togetherSnaewaert et al. The Flanders "acid beer" is fermented with cherries, and later on Lambic is added and the blend is allowed to referment in the bottle, creating something truly special (see reference <ref name="Verachtertsnauwaert"></ref>) looked at the microbial and metabolic composition in the finished beer of the same brewery as Martens et al., as well as two other Flanders red ale breweries using "state of the art" DNA sequencing methods. Three samples were analyzed (A, B, and C) from each brewery (1, 2, and 3). As expected, there were both similarities and differences between the three breweries tested, as well as some differences between the individual beers from each brewery.

====Modern AnalysisMicrobial Populations====A more recent study by Snaewaert et al. (see reference <ref name="snauwaert"></ref>) looked at the microbial and metabolic composition in the finished beer of the same Each brewery as Martens et al., as well as two other Flanders Red Ale breweries using "state of the art" DNA sequencing methods. Three had their three samples were analyzed (A, Bcompared, and C) from then also compared to each brewery (1, 2, and 3). As expected, there were both similarities and differences between the three breweries tested, as well as some differences between the individual beers from each breweryother. The bacterial populations for Breweries 1 and 3 were similar across all of their own samples, whereas Brewery 2's samples had a larger difference in between the three samples of its own beers. The yeast populations were similar between all samples for Brewery 1, whereas Breweries 2 and 3 had a larger difference in yeast populations between their three samples of beer <ref name="snauwaert"></ref>. In summary, Brewery 1 (presumed to be Rodenbach) had similar populations of bacteria and yeast across all samples of their beer, whereas Brewery 3 only had similar populations of bacteria and but not yeast across all samples of their beer, and Brewery 2 had the least amount of similarity in yeast and bacteria populations between their beer samples. This indicates that the beers from Brewery 2 were less consistent.

Overall, the samples were mostly dominated by ''Pediococcus damnosus'' and ''Brettanomyces bruxellensis'', except for two samples of Brewery 2 that were dominated by ''Acetobacter'' (smaller amounts of ''Acetobacter'' in Brewery 1 were identified as ''Acetobacter pasteurianus'', and might be the same species in Brewery 2), and one sample from Brewery 2 and two samples from Brewery 3 that were dominated by an unidentified yeast. Note that these findings conflicted with the findings of Martens et al. who identified ''P. parvulus'' as the dominating ''Pediococcus'' species. These two species are closely related, and an explanation of this discrepancy between the two findings was never found. ''Acetobacter'' numbers were higher in Brewery 1 and 2, and lower in Brewery 3, and were associated with the high acetic acid amounts found in the beers. Smaller amounts of ''Acetobacter'' in Brewery 1 were identified as ''Acetobacter pasteurianus'', and might be the same species in Breweries 2 and 3. Small numbers of ''Lactobacillus'' were found in all samples. ''Weissella'' and ''Leuconostoc'' were found in some samples from Brewery 2, whereas only trace amounts of ''Wessella'' were found in one samples from Brewery 3. A significant amount of ''Pichia'' yeast were found in two of Brewery 2 samples, and trace amounts in the third sample from that brewery. Only trace amounts of ''Pichia'' yeast strains were found in Breweries 1 and 3. Brewery 1 had some amount of ''Candida'' yeast in all samples. Brewery 3 had some amount of ''Candida'' in one sample, but only trace amounts in the other two samples, as well as and Brewery 2 had only trace amounts of ''Candida'' across all three samples. Traces of ''Kregervanrija'', ''Debaryomyces'', ''Priceomyces'', ''Hyphopichia'', and ''KregervanrijaWickerhamomyces'' yeastsconsisted of less than 0.01% of the DNA sequencing reads across all samples. Although detected with DNA sequencing methods, ''Lactobacillus'', ''Candida'', other yeast could not be isolated and grown under lab conditions <ref name="snauwaert"></ref>.

Snaewaert et al. also looked at the metabolic composition of finished beers in the three breweriesbefore they were packaged. Glucose was completely gone from the samples, but substantial concentrations of maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose were still present. This contrasts with the relatively high glucose and fructose found in finished bottled versions of these beers, which indicates that the beers are were back-sweetened with young beer or with residual sugar or even possibly just some form of sugar at bottling time <ref name="snauwaert"></ref>.

Overall the flavor of these beers was dominated by ethanol, lactic acid, acetic acid, ethyl acetate. Across the samples there was also a presence of isoamyl alcohol (31-150mg/L) and isoamyl acetate (1.99-6 mg/L), and an absence of 2-phenyl ethanol and 2-phenylethyl acetate in both the matured beers and the bottles versions of those beers. Small amounts of propionic acid, isobutyric acid, ethyl hexanoate, and ethyl octanoate were found. Higher levels of ethyl acetate were found compared to the Martens et al. study, and no ethyl decanoatewas found, which is a typical ester found in gueuze, was found. Just as the microbial populations of Brewery 2 differed from Breweries 1 and 3, so did it's its overall metabolite content. Higher levels of acetic acid were found in Brewery 2, which was attributed to high levels of ''Acetobacter'' populations. Additionally, all three of the samples from Brewery 2 differed from each other as far as metabolic content, which was also attributed to the microbial population differences between each of Brewery 2's samples <ref name="snauwaert"></ref>.