Pediococcus

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 , 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) . It is also seen as a major source of beer contamination in commercial breweries due to its ability to adapt to and survive in beer. 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 . Like many bacteria, Pediococci have the ability to transfer genes horizontally without reproduction . They are generally considered to be facultative anaerobes, which means they grow anaerobically but can also grow in the presence of oxygen . Some species/strains (including individual strains of P. damnosus) can have their growth and acid production inhibited by oxygen , while some will have better growth and produce more acid in the presence of oxygen (microaerophilic) . Strains found in beer are hop tolerant . 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 when exposed to a fresh sugar source. "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. It is considered a temporary flaw in sour beer. Some brewers, including Vinnie Cilurzo from Russian River Brewing and some Belgian lambic producers, claim that after the ropiness goes away (generally in 3-6 months ) it produces a deeper acidity and mouthfeel, and is viewed as a positive process in the production of sour beer . For other brewers, ropy beer is seen as a nuisance due to the beer needing to be aged for a longer period of time, especially when it occurs shortly after bottling. 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
Bootleg Biology	Sour Weapon (Pediococcus pentosaceus Blend)	P. pentosaceus blend	Perfect for acidifying unhopped wort quickly for kettle or “quick” sours. At 98F, it’s capable of achieving a pH of 3.3 within 18 hours. At 84F, it can reach a pH of 3.5 within 24 hours. With more time, a terminal pH of 3.1 may be reached. P. pentosaceus can also be used for long-term sours. It is capable of growing and producing lactic acid in worts with IBUs as high as 30, though it is recommended for unhopped worts as IBUs over 10 may prevent significant quick souring. At ~30 IBU, souring occurs in 2-3 months . This culture may produce antimicrobials called bacteriocins or pediocins. These can inhibit and kill similar species of bacteria like Lactobacillus and other Pediococcus species in mixed-culture fermentations. Read Bootleg Biology's Facebook post regarding bacteriocins for more info. No signs of ropiness (exopolysaccharides) have occurred in testing . It is still unknown how hops will affect souring in a long term scenario. Bootleg Biology is still researching long term effects and awaiting peoples feedback as of 5/23/2016.
Inland Island Yeast Laboratories	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. No longer available.
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.
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 . Homebrew vials contain 35 mL of slurry, and ~1.75-2.8 billion cells per vial .
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 . Homebrew packs contain 100-125 mL of slurry, and ~10-12.5 billion cells .

Manufacturer Tips

Bootleg Biology on Sour Weapon

Jeff reported good growth results using 2 grams of calcium carbonate (chalk; CaCO3) per liter of wort for starters of Sour Weapon . This same formula might benefit other Pediococcus starters as well. The CaCO3 serves as a buffering agent that keeps the starter pH from getting too low too fast, and is similar to the concept of buffered growth media for Lactobacillus (see Lactobacillus Starter).

Regarding exposure to oxygen in general:

"We don't propagate Sour Weapon on plates or at the liquid stage anaerobically, and definitely don't have problems with growth or souring. We have not tested pitching the culture in aerated wort, but there's certainly the chance that you could be increasing the likelihood of off flavors. Sour Weapon does still work very well when co-pitched with yeast in wort that has been aerated though. Hope that helps." - Jeff Mello of Bootleg Biology .

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 .

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 (~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 (~32 minutes in).

Metabolism

Lactic Acid Production

About 90% of sugar metabolized by Pediococcus produces lactic acid. It does so by homolactic fermentation producing primarily lactic acid (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) . However, all species in the Pediococcus genus are considered obligatory homofermentative because of the pathways that they use .

Malolactic Fermentation

See the Cider page.

Carbohydrate Metabolism

P. damnosus can ferment glucose, sucrose, and galactose. Some strains of P. damnosus can ferment maltose and sucrose . The disaccharide trehalose is the preferred carbon source for Pediococci . While simple sugars are the primary food source for Pediococcus, many strains of P. damnosus have been observed to produce varying degrees of both alpha and beta-glucosidase enzymes. Alpha-glucosidase enzymes have the ability to break down higher chain sugars, including dextrins, starches, and glucans (possibly even the glucans that are produced by P. damnosus that result in ropy beer). The types of beta-glucosidase enzymes produced by P. damnosus are thought to perhaps play a role in breaking down monoglycosidic bonds (see Glycosides), but cannot break down the more complex diglycosidic bonds which are needed to break down many glycosides that would release flavor and aroma compounds. Compared to the microbe Oenococcus oeni which is often used in wine and cider fermentation (malolactic fermentation) and has been shown to have more impactful beta-glucosidase activity, P. damnosus is thought to be less impactful on glycosides. Unlike O. oeni which decreases its enzymatic activity in low pH conditions, enzymatic activity of P. damnosus is very stable at a pH of 3-4. Very low concentrations of glucose or fructose (1 g/l) inhibit this enzymatic activity in P. damnosus. The presence of alcohol inhibits the alpha-glucosidase activity in most strains, which might contribute to longer lasting ropiness in beer. The optimal temperature for enzymatic activity in P. damnosus is between 35-40°C (95-104°F) .

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 . 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 after ~84 hours 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 4.3 billion cells/mL in MRS media starting at a pH of 5.5 , 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 .

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 . 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 . Therefore, it is generally best practice to seal the starter with an airlock.

Starter Information

General starter information for commercial Pediococcus cultures is limited. In addition to the various starter suggestions by various labs regarding their individual cultures, Jeff Mello of Bootleg Biology reported good growth results when using 2 grams of calcium carbonate (chalk; CaCO3) per liter of wort for starters of Sour Weapon . This same formula might benefit other Pediococcus starters as well. The CaCO3 serves as a buffering agent that keeps the starter pH from getting too low too fast, and is similar to the concept of buffered growth media for Lactobacillus (see Lactobacillus Starter). Starters of P. damnosus cultures could require around 84 hours for optimal growth, however this time requirement hasn't been looked at in wort starters (only MRS media) .

• For information on mixed culture starters, see Mixed Culture Starters.

"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

Some strains of P. damnosus can cause a beer (or wine) to go "ropy", also known as "sick" by lambic brewers (or more specifically as the fat sickness, “la maladie de la graisse” in French ). 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) . Some species of Pediococcus and other lactic acid bacteria have been reported to also be able to break down ropiness .

This "ropiness" is caused by production of exopolysaccharides (EPS) in the form of β-glucans (beta glucans) by some strains Pediococcus and some other lactic acid bacteria species. 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 . 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 , 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 .

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. Glucose 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 .

The presence of beta-glucans from barley have been observed to extend both the growth and the viability of Lactobacillus species in probiotics . One study looked at this effect in beta-glucans produced by Pediococcus parvulus and found that L. plantarum had a longer viability in a fermented medium with no additional food source when that medium was first fermented with P. parvulus and EPS was produced. The L. plantarum strain that was tested did not ferment the beta-glucans. This suggests that there is an interspecies simbiotic relationship between lactic acid bacteria that produce EPS and those that don't, and when EPS is produced (beta-glucans are present) the bacteria survive longer. The study also observed that more EPS was produced in an oat based wort and a rice based wort, while no EPS was produced in a barley based wort, suggesting that different food sources influence whether or not EPS is produced . No studies have been done on the growth or survivability of lactic acid bacteria in the present of EPS in beer, which is a more harsh environment than what was tested in these studies.

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

• Videos on MTF of ropy beer.

Exopolysaccharide pathway

Other Metabolites

P. damnosus can produce high amounts of diacetyl during lactic acid production . P. damnosus also produces an antimicrobial compound called pediocin PD-1, which can inhibit several bacteria spp including O. oeni . 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 .

Mixed Culture Influence

See Lactic Acid.

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 , and together with Lactobacillus are reported to be responsible for ~70% of all beer spoilage incidents caused by microbes . 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", "horC", and "horB" ). This has been thought to allow Pediococcus to adapt to living in beer .

While able to grow in the presence of hops, the presence of hops still inhibits P. damnosus. For example, one study found that in the presence of 15 IBU, lactic acid production was reduced by ~82%. Exposure to 15 IBU also increased diacetyl production by 350%, while 2,3-pentanedione (buttery, nutty, toasted, caramellic, diacetyl and acetoin notes ) was decreased by 25%. Interestingly, exposure to 3% ABV and no hops reduced the production of diacetyl and 2,3-pentanedione by about 20%. The addition of vitamins, specifically thiamine (vitamin B1) and riboflavin (vitamin B2), increased the production of lactic acid in the presence of 15 IBU or no hops by about 15-30%. However, thiamine also increased diacetyl production by 100-125% and 2,3-pentadione production by 20-30% without the presence of hops. In the presence of 15 IBU, thiamine and/or riboflavin increased diacetyl production by about 26-36% and 2,3-pentadione by about 40-114% . Strains that are grown exposed to small amounts of iso-alpha acids can be adapted to survive higher amounts up to ~150 μg mL¹. The implications of this in brewing mean that the bacteria can enter any stage of the process and remain in undetectable amounts until it adapts to higher IBU conditions and then potentially spoils beer .

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).

Justin Amaral recommends using isotonic sodium chloride and freezing media that is meant for 32°F freezers (lasts around 2 years) .

See Also

Additional Articles on MTF Wiki

• Alternative Bacteria Sources
• Spontaneous Fermentation
• Mixed Fermentation
• Mixed Cultures
• Scientific Publications
• Lactobacillus
• Glycosides

External Resources

• UC Davis article stub on Pediococcus damnosus.
• The Genera of Lactic Acid Bacteria, by W.H.N Holzapfel, Brian J.B. Wood. Partially free.
• MTF Thread with Bryan from Sui Generis on suggestions/theories on selecting for Pediococcus on plates.
• Dan Pixley's comparison between White Labs, Wyeast, and Inland Island Pediococcus cultures.