1. Cynthia M. Schultz BSc, MBA, PMP, Microbiologist
Quality Program Manager
Overview of Bacterial Contaminants/Microbial Load Management in Brewing
During the mashing of malt to create wort, the microbial load originating from the grain
diminishes greatly. Several thermotolerant (heat resistant) microbes - especially
homofermentative (ferment carbohydrates to lactic acid) lactic acid bacteria and some
types of bacillus may remain active in the moist nutritious environment. It is debatable
that some acid producing bacterial load may improve extraction, fermentability, and the
nitrogen yield of wort - plus enhance the foam stability, color, and flavor of the resulting
beer. The substantial beneficial effects of artificial mash acidification are achieved in
most breweries by direct acid addition or the utilization of acidulated malt. Additionally,
the suppression of other types of bacterial growth (non-acid producing) have been
shown to improve the filterability, extraction efficiency and nitrogen yield during the
mashing process - while fungal contaminants produce beta-glucanases and xylanases
lowering wort viscosity which improves mash filtration. Wort is a nutrient-rich, high-pH
(∼5.5) medium and the most historical prevalent spoilers were gram-negative
enterobacteria - especially species of Klebsiella, Citrobacter, Enterobacter,
Obesumbacterium and Escherichia. In wort, these bacteria produce DMS Dimethyl
Sulfide), organic acids, and 2-3-butanediol - giving beer an “off” character of a fruity or
vegetal aroma. In today’s brewhouses, these types of contaminants have been
significantly diminished due to GMP (Good Manufacturing Practices) and increased
sanitation protocols, incorporated with modern aseptic-style techniques.
Cool oxygenated wort undergoes the addition of Saccharomyces by the brewhouse to
begin the process of conversion from wort to beer. Fermentation of maltose/sugars
takes place producing ethanol and carbon dioxide. The resulting conditions created by
fermentation are hostile to the growth of most microorganisms and beer itself is high in
alcohol, carbon dioxide, contains hop-derived antimicrobial compounds, low in pH,
oxygen and mainly devoid of residual nutrients. 20% of all reducing sugars in “all-malt”
wort consist of oligosaccharides not utilized by Saccharomyces which is thought to be a
contributing factor to the mouthfeel and flavor of beer – although also have the potential
to support future microbial spoilage.
Gram-positive bacteria are found organically in plant matter (think barley/malt), humans
and generally in the environment. Fortunately, many Lactic Acid Bacteria are not able to
survive in beer due to the antibacterial activity of hop-derived compounds - although
there are some “super bug” forms of brewery contaminants that have adapted.
Microorganisms such as Pediococcus and Lactobacillus represent the greatest threat of
spoilage, as it is the most commonly reported contaminants of finished beers. Lactic
Acid Bacteria spoil beer through acidification, haze formation, and/or Diacetyl
production (buttery flavor). Many strains can also produce EPS (exopolysaccharides) in
beer, lending an oily consistency and occasional sliminess. Pediococcus species are

2. known for Diacetyl and EPS production due to their strong growth at low temperatures
and commonly contaminate both lagers and ales. Common Lactobacillus found in
breweries include; fermentum, buchneri, delbrueckii as well as other offenders such as
Pediococcus acidilacti, Leuconostoc lactis, and Lactococcus lactis. Early fermentation,
Enterobacteriaceae and Staphylococcus aureus may also occasionally be identified, but
they are unable to survive the fermentation process - mainly due to the low pH.
A major factor limiting which organisms can spoil beer (particularly alcohol and pH
tolerant gram-positive bacteria) is the involvement of hop-derived bittering compounds.
Hops impart many compounds that inhibit the growth of gram-positive bacteria, most
importantly the iso-alpha-acids assimilated from hop alpha-acid isomerization during
wort boiling. Iso-alpha-acids act as proton ionophores (allow protons to cross lipid
bilayers) disabling the transmembrane proton gradient (carries the electron transport
chain pump protons across the crista membrane) lowering cytoplasmic pH and halting
proton propulsion. Enzymatic activity and nutrient transport are diminished which
ceases cellular growth, ultimately killing the cell. Iso-alpha-acids, through cellular
reactivity mechanisms, cause high levels of cellular oxidative stress within the cells.
Hop-resistant HorA and beer-spoilage lactic acid bacteria differentiator ORF5, involve
several mechanisms for bacteriostatic (stops bacteria from reproducing) conditioning to
invoke hop resistant cellular responses. A key factor in hop resistance is the plasmid
encoded (and largely ATP dependent) transporter protein HorA, which rejects hop
compounds from within the cell. ORF5 is another plasmid encoded transporter has been
shown to allow for the facilitation of hop resistance across several species of Lactic Acid
Bacteria. In addition, those resistant cells upregulate (decrease receptors) the
expression of the hop-inducible cation (positively charged ion) within the HitA (hop-
inducible cation transporter), which may further facilitate manganese integration into
those hop-stressed cells. These complex responses created in resistant bacteria
indicate that hop-resistant Lactic Acid Bacteria are actually evolved to reproduce in beer
through acquired resistance to oxidative and acid stress. Kocuria kristinae (former
Micrococcus kristinae) and pH as well as Bacillaceae (Bacillus species) have low
potential of beer spoilage due to their acute sensitivity to hops, alcohol and lowered pH
environment. Four other species contain this hop resistance horA gene - Bacillus
cereus, Bacillus licheniformis, Staphylococcus epidermidis and Paenibacillus humicus.
It is believed throughout the scientific community that iso-alpha-acids have little impact
on the growth of gram-negative bacteria as well as yeast. As has been studied in
Saccharomyces cerevisiae, this non-growth phenomenon is due to relegation of iso-
alpha-acids within vacuoles, their active expulsion across cellular membranes and
adaptations in the cell wall itself in response to hop stress. In today’s brewery
environment, the main type of wild yeast contamination in beer is from spin-off strains of
Saccharomyces cerevisiae. These types of yeast spoil beer through ester and/or
phenolic off-flavor production, formation of haze, sediment and/or “superattenuation”
which can lead to overcarbonation and diminished body. In Saccharomyces and other

3. yeasts, phenolic off-flavor production is caused by p-coumaric acid and ferulic acid the
decarboxylation to 4-vinylphenol and 4-vinylguaiacol, facilitated by the POF1 (promoter
of filamentation) gene. These resulting compounds lend beer a medicinal or spicy clove
aroma - atypical component in most beer styles. Generally, any organism not
intentionally introduced is considered a spoilage organism, even yeast.
In conclusion, the two greatest risks to the overall microbial stability of beer are
pinpointed to the packaging and distributing of the final product. During the entire
modern brewing process, wort and beer are contained within sanitized and seamless
stainless steel vessels and travel within enclosed sterilized components - an excellent
mechanism to deter contamination and a large component of GBPs (Good Brewing
Practices). It is upon packaging that the beer comes in contact with several surfaces in
the filler and is even exposed shortly to air prior to final packaging. Breweries must be
diligent in ensuring biofilms are not forming within the filler unit or components within as
this increases the likelihood of biological contamination. Kegging can pose an even
greater risk as most breweries utilize kegs that are reused constantly and have interiors
with many complicated internal surfaces - making them difficult to sanitize completely.
These “rental” kegs (think Microstar) are circulated across many different breweries in
many different geographical locations repeatedly. Kegs undergo various undesirable
conditions such as intentional (and unintentional) exposure to such spoilage organisms
as Lactobacillus (often found in “sour” beers), Pediococcus, Brettanomyces as well as
countless others. These types of kegs undergo elevated and variable temperature
fluxes, take on oxygen, withstand uncontrolled humidity levels, and travel through many
types of external precipitation with almost constant movement across unknown and
undocumented environments.