Pediococcus

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Pediococcus - picture taken by Per Karlsson

Pediococcus (often referred to by brewers as "Pedio") are Gram-positive lactic acid bacteria (LAB) used in the production of Belgian style beers where additional acidity is desirable. They are native to plant material and fruits [1], and often found in spontaneously fermented beer as the primary source of lactic acid production (with P. damnosus being the only species identified in lambic) [2][3]. The ability to grow in beer is strain dependent rather than species dependent, however genetic differences indicate that P. damnosus and P. claussenii are better adapted to surviving in beer than P. pentosaceus [4]. Like many bacteria, Pediococci have the ability to transfer genes horizontally without reproduction [4]. They are generally considered to be facultative anaerobes, which means they grow anaerobically but can also grow in the presence of oxygen [5]. Some species/strains (including individual strains of P. damnosus) can have their growth and acid production inhibited by oxygen [6], while some will have better growth and produce more acid in the presence of oxygen (microaerophilic) [7][8]. Strains found in beer are hop tolerant [9]. Due to their continued metabolism of longer chain polysaccharides, acid production will increase with storage time. Pediococcus can form a pellicle.

Pediococcus may also cause “ropiness” (also called a "sick beer") due to the production of exopolysaccharides. "Ropy" or "sick" beer is more viscous and, in extreme circumstances, can form strands. Sickness effects mostly the mouthfeel and appearance of beer, and may have no influence on the flavor. Pediococcus species can also produce diacetyl with extended storage time. Brettanomyces can break down exopolysaccharides and diacetyl produced by Pediococcus and the two are often used together.

See Lactobacillus, Brettanomyces, Saccharomyces, and Mixed Cultures charts for other commercially available cultures.

Commercial Pediococcus Cultures

Name Mfg# Taxonomy Note
White Labs WLP661 damnosus High diacetyl (a.k.a. butter) producer and slow growing. Fermentation temp: 70-85°F (21-29.4° C). Attenuation: 65%. Cell count: 50-80 million cells/mL [10]. Homebrew vials contain 35 mL of slurry, and ~1.75-2.8 billion cells per vial [11].
Wyeast 5733 damnosus (previously listed as P. cerevisiae) May cause “ropiness” and produce low levels of diacetyl with extended storage time. Temp range: 60-95° F (15-35° C). Cell count: 1.0 x 108 (100 million) cells/mL [12]. Homebrew packs contain 100-125 mL of slurry, and ~10-12.5 billion cells [11].
RVA Yeast Labs RVA 601 damnosus Lactic acid bacteria used in souring Belgian-style beers such as gueze and Lambic. Acid production increases with storage. Temperature range is 60-95º F.
Inland Island Brewing & Consulting INISBC-998 damnosus Gram positive cocci that produces lactic acid. Also produces diacetyl and several proteins that may cause a "rope" to form in the beer. Rope will disappear with time. Oxygen and hop sensitive. 75-90°F Temperature Range.

Manufacturer Tips

Wyeast on 5733

"If using 1 pack of 5733 per 5 gallon batch; and either adding to secondary after alcoholic fermentation is complete, or co-inoculating with a Saccharomyces' strain, then a starter would not be necessary. If you did want or need to propagate, I'd recommend 2 liters of 1.030-1.040 wort per pack, incubated at 80-90*F, without agitation." - Michael Dawson, Wyeast.

"For propagation, we recommend using 1.040 OG wort and incubating at 30-35*C without aeration for 48-96 hours; pH drop will indicate when it's ready to pitch. For co-inoculation or post-primary addition, we recommend 0.5 million cells per mL, which is the equivalent of 1 pack in 5 gallons/20 liters. For larger volumes, you can propagate and inoculate with the starter culture at a rate 2.5-5% of the total volume of the main batch." - Michael Dawson, Wyeast.

White Labs on WLP661

"That one does well in 70-85 deg F. You can do a starter, but you shouldn't have to if you are doing a 5 gallon batch. It does take a while to sour, so just be patient and let it do it's thing." Sarah Neel, Sales and Customer Service, White Labs

Tips From MTF

The Rare Barrel

"We just performed an interesting experiment at The Rare Barrel with pedio. Jay, our head brewer and blender, wanted a more acidic beer that we could use as a blending component while also growing up our diminishing pedio culture. So we racked 2 oak barrels of gold fermented with Brettanomyces claussenii and White Labs Pediococcus damnosus (WLP661) into one of our 30 bbl batches of 12*p gold wort that had been acidified to about 4.5 pH in the kettle using lactic acid (our first hot side experiment!). No oxygen, really wanted to encourage the bacteria. Within 10 days the pH was 3.6 and the gravity 10.7. We were all surprised how quickly the pedio was working. We eventually racked a "splash" of fermenting gold with BSI Brett D and BSI Lacto D to drop the gravity. The beer had a bright acidity quickly and I was surprised at how well rounded the flavors were when we racked into barrels after a month in the fermenting vessel.

This might be a little harder to do at home, but I think there's potential for interesting results. Pediococcus shines long term traditionally so I agree with the posts above, if you're going for quick acidity I'd go lacto, but I plan on playing around with early Pediococcus fermentations at home." - Mike Makris from The Rare Barrel [13].

Pediococcus has a reputation for being a "slow worker", meaning that it produces acidity overtime. Staff at The Rare Barrel noticed that pitching on second generations of White Labs WLP661 Pediococcus might help the Pediococcus produce acidity very quickly [14] (~19 minutes in). They speculate that oxygen exposure might inhibit acid production with the White Labs WLP661 P. damnosus strain, however this is purely speculative and anecdotal [14] (~32 minutes in).

Metabolism

Lactic Acid Production

Pedio fermentables [15]

About 90% of sugar metabolized by Pediococcus produces lactic acid. It does so by homolactic fermentation (same EMP pathway as Lactobacillus homolactic fermentation), although some species/strains can convert glycerol to lactic acid, acetic acid, acetoin, and CO2 under aerobic conditions (P. damnosus is not in this category) [16]. P. damnosus can ferment glucose, sucrose, and galactose. Some strains of P. damnosus can ferment maltose and sucrose [1]. The disaccharide trehalose is the preferred carbon source for Pediococci [17].

Growth and Environment

P. damnosus is sensitive to temperature and pH. It is unable to grow at a pH of 8 or higher or at 35°C. The optimal growth occurs at 22°C and 5.5 pH. P. damnosus is sensitive to environments that contain NaCl, and will not grow with concentrations of 4% NaCl [1]. Most strains of P. claussenii can grow in beer. About half of the strains tested of P. damnosus can grow in beer, and none to very few strains of P. acidilactici, P. parvulus, and P. pentosaceus have been found to grow in finished beer.

One study showed that optimal growth was observed in MRS media with an initial pH of 6.7, and a final pH of 4.14, which occurred naturally from fermentation. The addition of bacteriological peptone, MnSO4, and Tween 80 also increased activity. Maximum cell densities of P. damnosus are around 1-2 billion cells/mL in MRS media of a pH of 6.7 (editor's note: this maximum cell density number needs to be verified) [18], but this is only in optimal conditions. Maximum cell density varies based on the conditions of the propagation with pH and nutrient demands being two of the main limiting factors [19].

Although more experiments are probably needed, agitation is believed to be an important factor for any species of microbe (yeast and bacteria). Gentle stirring on a stir plate or orbital shaker, or frequent gentle manual agitation leads to faster growth and a higher number of organisms. Agitation keeps the microbes in solution. It also maximizes the microbes' access to nutrients and disperses waste evenly. In a non-agitated starter, the microbes are limited to the diffusion rate of nutrients, leading to a slower and more stressful growth [20]. Although Pediococcus are generally considered facultative anaerobes and oxygen usually does not negatively affect their growth, some strains may show less growth in the presence of oxygen and are considered anaerophilic, meaning that the presence of oxygen inhibits their growth (and therefore their acid production) but they can still grow in the presence of O2. The presence of CO2 has a positive effect on acid production [6] . Therefore, it is generally best practice to seal the starter with an airlock.

"Ropy" or "Sick" Beer

Example of ropy beer brewed with The Yeast Bay Melange and dregs from Boon Mariage Parfait 2010. Photo is courtesy of Stuart Grant [21]

Some strains of P. damnosus can cause a beer (or wine) to go "ropy". Reportedly, ropiness in beer that also has Brettanomyces (which is traditionally credited with breaking down the ropiness after a period of rest) usually lasts anywhere from 1 week to 3 months, although fewer reports claim that it has lasted as long as 7 months (see reference for different experiences of brewers) [22]. This "ropiness" is actually caused by production of exopolysaccharides (EPS) in the form of β-glucans (beta glucans). A small amount of β-glucan is adequate enough to affect the visible viscosity of beer or wine. The gene known as "dps" has been identified with the production of β-glucan/EPS in P. damnosus, and the gene "gtf" in P. claussenii [4]. Not all strains of P. damnosus express the gene, and only ones that do will cause a beer to go ropy. Although it is not needed to survive in beer, EPS production is probably has importance in biofilm production [23], and Pediococci that are ropy have been found to be more acid, alcohol, and SO2 tolerant than other Pediococci. The thickness of the ropiness is increased with the presence of malic acid [24].

One study showed that the production of β-glucan coincided with the end of the growth phase of Pediococcus. While small amounts of β-glucan were produced during growth, after 2 days of growth, β-glucan production increased as growth slowed. β-glucan production stopped when growth stopped. This study showed that β-glucan production is linked to Pediococcous growth, producing more towards the end of growth. This would explain why beer containing Pediococcus often goes ropy shortly after naturally carbonating in the bottle. This study found that other variables were not factors in the production of β-glucan, such differing levels of alcohol (although alcohol interacts with the β-glucan in a way that makes the viscosity seem thicker). The study also found that the lack of agitation increased the β-glucan production (wine makers will often agitate or aerate ropy wine to cure the wine from ropiness). A higher initial pH encourages higher growth (5.5+), which increases β-glucan production. A lower initial pH (3.5), decreases growth and β-glucan production. A higher concentration of glucose increased growth and β-glucan production. A low starting pH decreases growth, and therefore decreases β-glucan production. Glocuse is needed for β-glucan production. While fructose alone is mostly insufficient, a combination of glucose and fructose was slightly more efficient than glucose alone [24].

It has been observed that Lactobacillus species can produce EPS (Lactococcus lactis, Lactobacillus delbrueckii, Lactobacillus casei, and Lactobacillus helveticus) [24].

Exopolysaccharide pathway [24]

Other Metabolites

P. damnosus can produce high amounts of diacetyl during lactic acid production [25][26]. P. damnosus also produces an antimicrobial compound called pediocin PD-1, which can inhibit several bacteria spp including O. oeni [27][28]. P. claussenii tends to produce a smaller amount of acetic acid than lactic acid in about a 1:3 ratio. P. damnosus tends to produce only lactic acid and no acetic acid [17].

Mixed Culture Influence

Editor's note: special thanks to Richard Preiss of Escarpment Laboratories for helping to interpret the science referenced this section.

P. damnosus, as well as some other bacteria, have been shown to alter the expression of genes in most, but not all, Saccharomyces cerevisiae strains (and other Saccharomyces species and even perhaps Brettanomyces), in a way that changes how they ferment sugars, and essentially forms a symbiotic environment with the yeast. Normally Saccharomyces will only ferment glucose when glucose is present and ignores other sugars such as maltose and maltotriose until the glucose is gone. Biologically speaking, when the presence of glucose in the yeast's environment shuts down the yeast's ability to ferment any other types of sugar besides glucose, this is called "glucose repression" [29]. Saccharomyces and Brettanomyces bruxellensis (it is currently not known if other Brett species other than B. bruxellensis have this ability) have a gene called "GAF+" that when expressed actually allows it to bypass this "glucose repression" and ferment the other sugars simultaneously. Normally this gene is not expressed except by a very small number of cells [30].

When P. damnosus lives together with Saccharomyces, a chemical is produced by P. damnosus that essentially "turns on" the GAF+ gene in Saccharomyces. Not only does Saccharomyces then have the ability to ferment other sugars at the same time as glucose, but it produces less alcohol. Viability over time is also increased in Saccharomyces cells that express this gene versus those that don't. In wild fermentation of grapes, the wild GAF+ Saccharomyces strains thrived over the other types of fungi that were found on the wild grapes. This led the researchers of the referenced study to speculate that the GAF+ gene may play a role in preventing other fungi from thriving. It is thought that this benefits both microorganisms, which are often found together in the wild during fermentation of fruit; the bacteria isn't killed by higher alcohol levels, and the yeast has a broader food source. Furthermore, once a Saccharomyces cell expresses the gene, it will continue to pass this gene expression onto its offspring. In winemaking, this is the cause of arrested wine fermentations due to the lower amount of alcohol produced. For example in the referenced study, the GAF- Saccharomyces cells fermented grape must into a 12% ABV wine, and the GAF+ Saccharomyces cells fermented the same wine must into an 8% ABV wine [29]. However, the implications of this in sour beer brewing are much different and have yet to be explored scientifically.

Of all the Lactobacillus species that have been studied for this behavior (L. brevis, L. hilgardii, L. plantarum, and L. kunkeei), only L. kunkeei was shown to induce the GAF+ gene. Some species of bacteria from the genres Staphylococcus, Micrococcus, Bacillus, Listeria, Paenibacillus, Gluconobacter, Sinorhizobium, Escherichia, Serriatia, and all Pediococcus species tested also influenced the GAF+ gene in Saccharomyces [29].

Hop Resistance

Pediococcus species and strains are generally resistant to hop compounds, and have been reported to grow in beer with at least 30 IBU [17]. It has been suggested by research that horizontal gene transfer (transfer of genetic material by means other than reproduction) allows Pediococcus species (and other LAB) to obtain the genes associated with resistance to hops (primarily multi-drug transporter "horA", along with "hitA" or "horC"). This has been thought to allow Pediococcus to adapt to living in beer [4].

Storage

For instructions on how to make slants at home capable of storing any microbe for potentially 2+ years, see Bryan's video on Sui Generis Brewing (requires a pressure cooker).

See Also

Additional Articles on MTF Wiki

External Resources

References

  1. ↑ 1.0 1.1 1.2 Viticulture & Enology. UC Davis website. Pedioccous damnosus. Retreived 07/28/2015.
  2. ↑ The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer. Freek Spitaels, Anneleen D. Wieme, Maarten Janssens, Maarten Aerts, Heide-Marie Daniel, Anita Van Landschoot, Luc De Vuyst, Peter Vandamme. April 18, 2014.
  3. ↑ Multiple Scientific publications linked on MTF.
  4. ↑ 4.0 4.1 4.2 4.3 Comparative genome analysis of Pediococcus damnosus LMG 28219, a strain well-adapted to the beer environment. Isabel Snauwaert, Pieter Stragier, Luc De Vuyst and Peter Vandamme. 2015.
  5. ↑ Lactic Acid Bacteria. Todar's Online Texbook of Bacteriology. Kenneth Todar, PhD. Pg 1. Retrieved 08/09/2015.
  6. ↑ 6.0 6.1 TAXONOMIC STUDIES ON THE GENUS PEDIOCOCCUS. ATSUSHI NAKAGAWA, KAKUO KITAHARA. 1959.
  7. ↑ THE NUTRITION AND PHYSIOLOGY OF THE GENUS PEDIOCOCCUS. Erling M. Jensen and Harry W. Seeley. 1954.
  8. ↑ Pediocins: The bacteriocins of Pediococci. Sources, production, properties and applications. Maria Papagianni and Sofia Anastasiadou. 2009.
  9. ↑ Comparative genome analysis of Pediococcus damnosus LMG 28219, a strain well-adapted to the beer environment. Isabel Snauwaert, Pieter Stragier, Luc De Vuyst and Peter Vandamme. April 2015.
  10. ↑ Private correspondence with White Labs Customer Service and Dan Pixley. 10/29/2015.
  11. ↑ 11.0 11.1 Conversation with Richard Priess regarding WL and Wyeast homebrew pitches. 10/30/2015.
  12. ↑ Wyeast Specifications 2015 Retail Products. 2015.
  13. ↑ Conversation with Mike Makris on Milk The Funk.
  14. ↑ 14.0 14.1 The Sour Hour Episode 29 on The Brewing Network, interview with The Rare Barrel staff. 02/19/2016.
  15. ↑ Wine Microbiology. Practical Applications and Procedures. Kenneth C. Fugelsang, Charles G. Edwards.
  16. ↑ Encyclopedia of Food Microbiology. Pediococcus. Carl A. Batt. Academic Press, Sep 28, 1999 .
  17. ↑ 17.0 17.1 17.2 Metabolic strategies of beer spoilage lactic acid bacteria in beer. Andreas J. Geissler, Jürgen Behr, Kristina von Kamp, Rudi F. Vogel. 2015.
  18. ↑ Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1. Nel HA, Bauer R, Vandamme EJ, Dicks LM. 2001.
  19. ↑ Neva Parker, Reddit thread. 10/29/2015.
  20. ↑ Conversation with Bryan of Sui Generis Blog about starters and agitation. 11/09/2015.
  21. ↑ Conversation with Stuart Grant on MTF. 09/16/2015.
  22. ↑ Poll on Milk The Funk regarding how long ropy beer has been observed. 08/20/2015.
  23. ↑ Ethanol tolerance of lactic acid bacteria, including relevance of the exopolysaccharide gene gtf. Pittet V, Morrow K, Ziola B. 2011.
  24. ↑ 24.0 24.1 24.2 24.3 Glucose fermentation kinetics and exopolysaccharide production by ropy Pediococcus damnosus IOEB8801. Emilie Walling, Marguerite Dols-Lafargue, Aline Lonvaud-Funel. Food Microbiology Volume 22, Issue 1, January 2005, Pages 71–78.
  25. ↑ Identification of pediococci by ribotyping. R. Satokari, T. Mattila-Sandholm and M.L. Suihko. Journal of Applied Microbiology 2000, 88, 260–265.
  26. ↑ The Microbiology of Malting and Brewing. Nicholas A. Bokulicha, and Charles W. Bamforth. June 2013.
  27. ↑ Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1. H.A. Nel1, R. Bauer, E.J. Vandamme and L.M.T. Dicks. Jan 2002.
  28. ↑ Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832. Bauer R, Chikindas ML, Dicks LM. May 2005.
  29. ↑ 29.0 29.1 29.2 Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of Metabolism. 2014. Daniel F. Jarosz, Jessica C.S. Brown, Gordon A. Walker, Manoshi S. Datta, W. Lloyd Ung, Alex K. Lancaster, Assaf Rotem, Amelia Chang, Gregory A. Newby,David A. Weitz, Linda F. Bisson, and Susan Lindquist. Cell. 2014 Aug 28;158(5):1083-93.
  30. ↑ An Evolutionarily Conserved Prion-like Element Converts Wild Fungi from Metabolic Specialists to Generalists. Daniel F. Jarosz, Alex K. Lancaster, Jessica C.S. Brown, Susan Lindquist. Cell. Volume 158, Issue 5, p1072–1082, 28 August 2014
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