Nebraska Grower and Brewery Conference

January 5 - 6, 2017

Microorganisms in the brewery:

From Acetobacter to Zymomonas and
most everything in-between
Bob Hutkins
The beer microbiome
The brewery microbiome
For microbiologists, each brew
is a microbiology experiment
1. Medium preparation
2. Inoculation and incubation
3. Separate organisms from medium
4. Aseptic packaging
Why brewers should care about
brewery microorganisms:

1. Safety
2. Compliance
3. Quality
4. Opportunity
 Safety
Manufacturing
Accidents
Physical, chemical and biological hazards

Product
Allergens
Microbiological
 Compliance
GMPs, HACCP, FDA, and FSMA
All food producers must register with the FDA
This includes breweries
 Compliance
GMPs, HACCP, FDA, and FSMA
All food producers must register with the FDA
This includes breweries
This means that breweries must have GMPs
FSMA is also headed your way
 Good Manufacturing Practices
(GMPs)

Good Manufacturing Practices for Craft Brewers

(GMPCB), developed by the Brewers Association)

GMPs consists of SOPs and other minimum

requirements that all food manufacturers must meet to
assure products are of high quality and do not pose any
risk to the consumer or public.

GMPs are prerequisite for HACCP

GMP compliance is required (or else)

 Good Manufacturing Practices
in the Brewery
Why?
ensure a clean and safe environment for employees
ensure products are safe
ensure sanitary conditions are in place.
reduce risks associated with pest infestations
reduce risks associated with public health hazards
reduce all other risks that could harm a business
(including being shut down or action by civil suits)
 Good Manufacturing Practices
in the Brewery
Why not?
ensure a clean and safe environment for employees
ensure products are safe
ensure sanitary conditions are in place.
reduce risks associated with pest infestations
reduce risks associated with public health hazards
reduce all other risks that could harm a business
(including being shut down or action by civil suits)
Implementing Good Manufacturing Practices

Use quality raw materials

Perform lab tests that can be validated and/or trusted
Establish SOPs
Educate and train staff
Recognize and account for deviations in product quality
Create a good quality management system
Package and label the product according to guidelines
Meet (or exceed) sanitary and processing requirements
 The beer and brewery microbiome
Who is there, from where do they come,
and what are they doing?
 The beer and brewery microbiome
1. Who is there?
Saccharomyces Brettanomyces
yeast non-Saccharomyces Kloeckera
Pichia

Lactobacillus
lactic acid bacteria Pediococcus

acetic acid bacteria Enterobacteriacae

Staphylococci wild yeast and fungi Zymomonas

 Lactic acid bacteria
A B C

5.0 µm

D E D

A, Lactobacillus delbrueckii; B. Lactobacillus brevis; C, Pediococcus

pentosaceus; D, Pediococcus damnosus; E, Lactobacillus helveticus
Genera of lactic acid bacteria
Aerococcus
Carnobacterium
Enterococcus
Lactobacillus
Lactococcus
Leuconostoc
Oenococcus
Pediococcus
Streptococcus
Tetragenococcus
Vagococcus
Weissella
Lactic acid bacteria important in fermented foods

Lactobacillus
Lactococcus
Leuconostoc
Oenococcus
Pediococcus
Streptococcus
Tetragenococcus
Lactic acid bacteria important in beer

Lactobacillus
Pediococcus
Diagnostic characteristics of lactic acid bacteria

Fermentative
Low mol% G + C
Non-sporeforming
Gram positive rods and cocci
Facultative anaerobes
Catalase negative
Non-motile
Acid-tolerant
Practical characteristics of beer LAB

Homofermentative (e.g., Pediococcus damnosus)

Heterofermentative (e.g., Lactobacillus brevis)
Hop tolerance
Ethanol tolerance
Produce diacetyl
Produce biofilms
Produce biogenic amines
 The beer and brewery microbiome
2. From where do they come?

malt water adjuncts

ingredients hops yeast flavors

environment air, water

equipment kettles, fermenters, tanks, fillers

personnel worker hands, clothing

The brewery microbiome
Source of microorganisms in the brewery
Ingredients

LAB, fungi important during

barley malt steeping and germination

Fusarium important for antimicrobial

hops activity, not as a source of m/o
toxins gushing
source of starter strains, but
yeast also contaminants

water
air/gas
Source of microorganisms in the brewery
Equipment

milling, mashing, lauter tun

kettle and whirlpool separator

fermentor

maturation, bright tanks

packaging
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Potential sources of microbiological contamination (!) during the

brewing process.
Vaughan et al., 2005. J. Inst. Brew. 111:355–371.
 Abundance of micoorganisms in the brewery

Source: Bokulich et al. eLife 2015;4:e04634

 Distribution of bacteria in the brewery

Source: Bokulich et al. eLife 2015;4:e04634

 The beer and brewery microbiome
3. What are they doing?
yeasts
anaerobic
maltose, other sugars ethanol + CO2
fermentation
glycerol
diacetyl butanediol
phenolics
acetic acid
acetaldehyde
esters, higher alcohols

amino acids H2S, SO2, other sulfites

 The beer and brewery microbiome
3b. What are they doing?
bacteria
anaerobic
maltose, other sugars lactic acid
fermentation
LAB
diacetyl

aerobic
acetic acid AAB
oxidation

amino acids biogenic amines LAB

Microbiological quality control in
the brewery

Same principles apply for beer as for any other

perishable food

Thus, for beer (and other fermented foods), it’s a

very thin line between fermented and spoiled
Spoilage versus Success:
a matter of control

Exert control by:

Acidity and pH
Temperature
Moisture
Salt
Substrate availability
In beer, control is by:
Low pH
Temperature
Ethanol
Hops
Absence of oxygen
Substrate availability
Beer spoilage should be rare, because:
low pH, high ethanol, iso-alpha acids
beer is attenuated, few nutrients
anaerobic, aerobes cannot grow
kettle boil (kill step)
stainless steel everywhere

But: acid-, ethanol-, and hop-tolerant organisms

exist in nature, including breweries
wort is nutrient-rich
exposure not insignificant (with plenty of
opportunities for contamination
 Beer spoilage organisms: from field to glass
Adapted from Vaughan et al., 2005. J. Inst. Brew. 111:355–371.

barley in field/ Aspergillus

malting Fusarium

mashing/ Pediocuccus Klebsiella

wort separation Bacillus Citrobacter

Pediococcus Zymophilus
fermentation
Selenomonas Obesumbacterium

stabilization/ Pediocuccus Pectinatus

packaging Lactobacillus Megasphaera

Zymomonas Pectinatus Pediocuccus finished beer

Micrococcus Megasphaera Lactobacillus

Acetobacter Pediocuccus dispense

Gluconacetobacter Lactobacillus
 Controlling microbial defects in beer
defect cause control
diacetyl Pedicococcus cleaning
sanitation
Lactobacillus GMP
cleaning
lactic acid lactic acid bacteria sanitation
(LAB) GMP
cleaning
acetic acid acetic acid bacteria sanitation
(AAB) GMP
cleaning
sulfur LAB, AAB, others sanitation
GMP
cleaning
haze LAB, yeasts sanitation
GMP
Other strategies to prevent microbial defects

acid addition
mash acidification lactic acid fermentation
 Viable counts of lactic acid bacteria during a typical lager mash

LAB cfu
per ml

Vaughan et al., 2005. J. Inst. Brew. 111:355–371.

 Other strategies to prevent microbial defects

acid addition
mash acidification lactic acid fermentation

fermentation alpha acid-rich hops

pasteurization
packaging/distribution filtration
There are always “new” trouble-makers
Pectinatus and Megasphaera
strict anaerobes
offensive rotten egg aroma
extensive turbidity
problematic in late stages of brewing

“Hard-to-culture” LAB
Viable But Not Culturable (VBNC)
Hop-resistant LAB
Strategies for preventing microbial spoilage
(works for food, beer, anything)

1. Keep out (asepsis)

2. Remove them

3. Prevent/delay growth

4. Kill/destroy

5. Combinations (Hurdles)
 Hurdle Concept in Beer

organism A
organism B

ethanol hops pH Eh T
 Synergistic Effect of Hurdles

ethanol hops pH Eh T

ethanol hops pH Eh T
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Filler

Potential sources of microbiological contamination (!) during the

brewing process.
Vaughan et al., 2005. J. Inst. Brew. 111:355–371.
Contamination hotspots:
Fitting into tight spots
Beer

Bacteria
LAB
AAB

Yeast
 The filling station is critically important, as this is
where beer is especially vulnerable to contamination

This short gap, both in space

and time, is sufficient for all sorts
of organisms to gain access
Surveying for microbes in the brewery

1. Microscopic

2. Cultural

3. Indirect

4. Molecular
 Microscopy for counting:
Easy, informative, inexpensive
 Cultural
Can be qualitative or quantitative
Can be general purpose or group-specific
These depend on the methods and media
Selective v. Non-selective
Detection v. Enumeration
Detection v. Identification

MRS UBA
 Traditional Methods

Also informative, but can take 3 – 5 days

 Rapid, qualitative tests
ATP Luminescence

Can be used for rinse water or equipment (swabs)

Easy, rapid, informative

Nearly real-time

Relative Light Units (RLU) can be scored as pass/fail

Source: Hill, A. 2015. Brewing Microbiology: Managing Microbes, Ensuring Quality and Valorising Waste
Modern methods
ID to species level
 How to know, before it’s too late,
that you have a problem?

This is the key reason for having a HACCP

plan.

HACCP, after all, is intended to prevent

problems

Thus, regular assessment of ingredients,

equipment, and processing is the best way
to minimize risks of contamination
 Consider the following scenarios:
What is the tolerance for a mistake?

Soldiers deployed to Afghanistan with three week rations

Shuttle astronauts at the Space Station for 3 months

Caterer to Olympic Athletes in Rio

Infant food manufacturer

New craft brewer with investors

Common goal = zero defects
 Sanitation basics
“brewing = 90% cleaning + 10% paperwork”

HACCP
Hygiene in the brewery
choice of cleaners and sanitizers
dealing with biofilms and chronic problems
Assessment and testing
 Biofilms in the brewery
Biofilms can form on equipment, even stainless steel
Bacteria within biofilms are more resistant to heat,
antimicrobials, and cleaning and sanitizing agents
Biofilms will continue to shed bacteria into the food
Lambic, coolhouse and sour beers
Perhaps the original beer produced by humans
Initiated by spontaneous or wild fermentation
In Belgium, lambics are seasonal, fall to spring
This is because the boiled wort needs to cool
overnight to about 70 F in a shallow open vessel
called a “koelschip”
The U.S. version, called “coolship ale”, mimics the
traditional lambic process
Microbiology of lambic-style beers
Fermentation is a classic ecological “succession”
One group makes conditions right for the next
Four general distinct, but overlapping phases:

1. Enterobacteriacae
2. Primary alcoholic fermentation
3. Acid fermentation
2. Attenuation
 ACA fermentation profile

yeast

bacteria

pH

plato

Bokulich et al., 2012. PLOS ONE

 Bacterial diversity of traditional spontaneously fermented lambic beer

1 2 3 4

Spitaels et al., 2014. PLOS ONE

 Yeast diversity of traditional spontaneously fermented lambic beer
1 2 3 4

Spitaels et al., 2014. PLOS ONE

 Brewhouse microbiota are responsible for fermentation of American Coolship Ale

Bokulich et al., 2012. PLOS ONE

Questions