Strictly anaerobic beer-spoilage bacteria

with special emphasis on new and

emerging species

The 26th Nordic Meeting on Brewing Technology

Riikka Juvonen
VTT Technical Research Centre of Finland
 27/09/2012 2

What are strictly anaerobic beer-spoilage bacteria?

 Need oxygen-free, i.e. anaerobic, environment

for growth

 Spoil packaged beers

 turbidity and rotten egg-like and rancid
off-flavors
 great economic losses
 Found in the late 70s -> the most recent group
of beer spoilers

 Increasing number of new species

 27/09/2012 3

“Old” strictly anaerobic beer-spoilage species

 LATE 70´s
 Pectinatus; combed bacteria
 P. cerevisiiphilus; beer lover
 P. frisingensis (named 1990); from Freising
P. frisingensis
 Megasphaera; big sphere
 M. cerevisiae; of beer

 EARLY 80´s (named 1990)

 Selenomonas; crescent shaped bacteria M. cerevisiae
 S. lacticifex ; a maker of lactic acid
 Zymophilus; yeast lover
 Z. raffinosivorans; raffinose devouring
 Z. paucivorans ; uses few carbohydrates
S. lacticifex
 27/09/2012 4

New and possibly emerging strictly anaerobic

beer-spoilage species

NEW BEER SPOILERS IN 2006:

 Pectinatus haikarae (1988), in honor of doc. Auli
Haikara
 Megasphaera sueciensis (1997), of Swedish origin
 Megasphaera paucivorans (2003), uses a few
substrates

NEW POSSIBLY EMERGING SPOILERS IN 2004-2012:

 Pectinatus portalensis (2004); from El Portal

 Pectinatus brassicae (2011); of cabbage

 ”Pectinatus sottaceto ”(2012); in honor of USDA

Agricultural Researc Service Images: Juvonen 2009,
Caldwell et al. 2012
 27/09/2012 5

Where are the ”old species” found?

• Steeping water of malt before milling
• Water pipe systems
Selenomonas • Pitching yeast
Zymophilus • Main and secondary fermentations
(Pectinatus) • The CO2 membrane and lines
(Megasphaera) • Washed bottles
• Drainage
MAIN
SECONDARY • Water from the floor and ceiling of
FERMEN-
FERMEN- filling hall
WORT TATION
TATION • Air in filling hall
• Rinse water, biofilm samples, and
CO2 channel in filling lines
• Conveyor belt lubricant
• Finished beer

PRESSURE
TANK

FILTRA - FLASH PASTEURISATION FILLING Bottled

TION OR STERILE FILTRATION
beer
Pectinatus, Megasphaera
 27/09/2012 6

Comparative DNA analysis reveals new

habitats for Zymophilus and S. lacticifex

 Zymophilus species are closely related

to a tree pathogen (Propionispira)
 plant-associated in nature
 to breweries with malt and hops

 S. lacticifex Image: Tree affected by bacterial

wetwood disease
 biohydrogen production from
anaerobic sludge
 wastewater

Image: Biohydrogen production

system
 27/09/2012 7

Where are the ”new” and emerging species found?

Species Geography Sources

P. haikarae Germany, Finland, Spoiled beer, air in bottling hall,
Sweden, Norway beer filling line
M. paucivorans Italy, UK2, China2, Spoiled beer, beer filling line2
Uruguay2 Biohydrogen production using anaerobic
sludge and cheese whey*
M. sueciensis Sweden, UK*, ”
China*, Uruguay*
P. portalensis Spain, Holland, Winery wastewater, biohydrogen production using
Uruguay cheese whey, fermentation of distillery wastewater
P. brassicae China Pickled cabbage wastewater

P. sottaceto1 USA Spoiled cucumber fermentation

*M. sueciensis and M. paucivorans were not discriminated

References: Juvonen & Suihko 2006, Castello et al. 2009, Sun et al. 2010, Paradh et al. 2011, Ning et al. 2012,
Voetz, 2010, Gonzalez et al. 2004, Temudo et al. 2008, Chang et al. 2011, Caldwell et al. 2012 (submitted)
 27/09/2012 8

Occurrence of the strictly anaerobic beer spoilers

in the major UK breweries

Species Brewery
1 2 3 4 5 6 7 8 9 10
Samples 10 10 10 10 15 15 7 10 10 20
P. cerevisiiphilus 1 1
P. frisingensis 2 2
P. haikarae
M. cerevisiae 1 1
M. paucivorans & 1
M. sueciensis
Sampling time March-August September-February

A. D. Paradh, W. J. Mitchell and A. E. Hill, J. Inst. Brew. 117(4), 498–506, 2011

 27/09/2012 9

How beer gets contaminated?

 Usually enter beer during filling operations -> sporadic

 Filling lines prone to biofilm formation

 Sewer system probably an important reservoir

 Spreading via aerosols

Biofilm on stainless
 Contamination of the whole process also possible -> steel brewery
equipment
high frequency Image: Outi Priha
 27/09/2012 10

Effects in beer production

Effects on Effects in finished beer Off-flavor

Genus fermentation Turbidity Metabolites

Acetic, propionic, butyric, Rancid,

Megasphaera1 unknown + caproic, isobutyric, isovaleric
and valeric acids, H2S rotten egg

Acetic, propionic, (succinic

Pectinatus1 possible and lactic) acids, acetoin,
+ Rotten egg
inhibition H2S, organic sulphur
compounds
Selenomonas unknown + Lactic, acetic, and propionic
Sour
acids

Zymophilus 2 unknown + Acetic and propionic acids

Sour
(lactic acid)
1 Beer-spoilage ability is considered univeral property shared by all strains
2 Atelevated pH of 5–6
3 Foaming, only slight odour in case of P. haikarae
 27/09/2012 11

Properties related to beer spoilage

The ”old” beer-spoilage species

Pectinatus, M. cerevisiae – obligate spoilers

 acid-tolerant: P. frisingensis (pH <4) > M. cerevisiae (pH 4.0-4.1)
 ethanol tolerant: P. frisingensis (~5.2%, w/v) > M. cerevisiae (<4.2%)
 hop-tolerant (33-38 IBU)
 growt at low temperatures (8-15 °C)
 good oxygen tolerance at low temperatures (Doxy >> 2 days)
 growth at 0.96-1.9 mg/l of dissolved oxygen

Zymophilus, S. lacticifex – potential spoilers

 Zymophilus species grow at pH 5-6
 S. lacticifex grows at pH 4.3-4.6
 27/09/2012 12

Properties related to beer spoilage

M. cerevisiae survives well in water and beer at low temperatures

Growth of stressed and non-stressed M. cerevisiae in beer with 50% MRS medium after a) 0-, 3- and
7-day storage in water at 13oC, and b) 0-, 3- and 7-day storage in beer at 7oC
 27/09/2012 13

Spoilage ability of Pectinatus frisingensis

in traditional and novel types of soft drinks
1,0E+07
2d 1 mth 3 mth = Growth in encrichment
broths
1,0E+06

1,0E+05
Inoculum level
cfu / ml

1,0E+04

1,0E+03

1,0E+02

1,0E+01
 27/09/2012 14

Properties related to beer spoilage

New beer-spoilage species
 P. haikarae
 spoilage incidents only in low-alcohol beers
 acid tolerance similar with P. cerevisiiphilus
 physiology adapted to brewing environment
 grows at ≤15°C
 able to detoxify oxygen radicals

 Megasphaera spp.
 M. paucivorans grew in beer with 5 vol-% ethanol, pH 4.3
 M. sueciensis grew in beer with 2.8 vol-% ethanol, pH 4.9
 grow well at 15 °C
 puryvic and gluconic acids preferred energy sources
 27/09/2012 15

Properties related to beer spoilage

Emerging beer-spoilage species

 P. portalensis is a potential spoiler of beers and

wines with up to 15% alcohol

 P. sottaceto is a potential beer spoiler

 light beer, 4 vol-% alc., ca. 10 BU, pH 4.2

 P. brassicae acid-tolerant enough (pH 3.5) to

grow in beer
 no information about beer-spoilage ability
 27/09/2012 16

How to detect strict anaerobes in breweries?

Cultivation methods
BEER
 Avoid exposure to air BEER+nutrients

 Forcing tests the only widely applicable method

2-6 weeks
 Many common media, e.g. MRS, NBB-B and
PYF broth, suitable
SENSORY
 A selective medium for Megasphaera and CHANGES - smell,
Pectinatus (SMMP) (Lee 1994) turbidity, color

 Confirmation by smell and microscopy

 The same media and methods also suitable for

the new Megasphaera and Pectinatus species

Gram-negative cocci or
motile ”snake-like” rods
 27/09/2012 17

How to detect strict anaerobes in breweries?

New cultivation methods

 EnBase Growth System based media

(BioSilta Oy, Finland)

 Selective plate medium for anaerobic

bacteria in beer samples

 Slow enzymatic release of glucose

 Preliminary results with Pectinatus in

beer promising
 stressed cells (~50 cfu/100 ml)
 aerobic membrane filtration Juvonen & Storgårds, VTT, unpublished results
 anaerobic incubation at +26°C
 visible colonies after 4 d Excellent recovery despite of
aerobic filtration step
 27/09/2012 18

How to detect strict anaerobes in breweries?

New cultivation methods

Composition of MRS-T:
 Modified MRS medium (MRS-T) for
selective detection of Pectinatus Component Amount
MRS broth Normal
 Hygiene samples and spoiled beer Sodium thioglycolate 0.25 g
Cysteine hydrochloride 0.25 g
Tetrahydroiso-α-acids 50 mg
 Semisolid agar in closed tubes
2-Phenylethanol 0.15%
Agar bacteriological 0.2%
 Simple preparation and use Distilled water 1,000 mL
Final pH 6.5
 Incubation 28°C 1-2 days
Matoulkova et al. 2012, J. Am. Soc. Brew.
Chem. 70(1):29-34, 2012
 Confirmation by microscopy
 27/09/2012 19

Alternative / supplementary methods

Principles

 Chemical analyses – detection of metabolites, cell components

 Imaging techniques– visualization of cells / microcolonies

 Immunoassays – fluorescent antibodies for cell antigens

 Nucleic acid based methods – detection of DNA or RNA

 Amplification of signature DNA segments
 PCR, LAMP
 Hybridization with labeled DNA probes
 FISH (fluorescence in situ hybridization) – RNA detection in cells
 Microarrays for detection of labeled RNA
 Hybridization protection Assay (HPA) for RNA detection
 Sandwich hybridization of rRNA and DNA probes on beads
 27/09/2012 20

Detection methods for strictly anaerobic beer-spoilage species

Detection Detection Identifi- Detecting Diagnostic Species
limit time* cation dead cells kits (costs)
Cultivation 1 cfu 2-6 weeks No No No All
< 1 week (ca. 1 €)
Chemical 105-106 Saves Coarse Yes No All
detection cells/ml 1-2 d
DEFT 103 cells 0.5-1 h No Yes/No No All
Microcolony 1 cfu 2d No No No
Immuno- 20-40 3h Yes Yes No Pectinatus,
assays cells/10 ml M. cerevisiae
PCR 101-103 2-8 h* Yes Yes Yes Group, genus,
cells (12 €) species
Sandwich 105-106 3 h* Yes No Yes P. frisingensis,
hybridization cells/ml (3 €) P. cerevisiiphilus
FISH 103 cells 3-5 h* Yes No Yes Not P. haikarae
(15 €)
HPA 103 cells 1.5 h* Yes No No Pectinatus spp.

* In general 1.4 days of enrichment is needed to detect trace contamination

 27/09/2012 21

Identification and tracing of contamination routes

using DNA-based methods

 16S rRNA gene sequencing (Juvonen & Suihko, 2006)

 Reliable identification except M. paucivorans/sueciensis

 DNA fingerprinting techniques

 Automated ribotyping (Suihko & Haikara 2001)
 Species identification
 Tracing of contaminations, reoccurrence
 Fingerpint libraries at VTT (public, custom)

 PCR-RFLP analysis (Juvonen et al. 2008, Ohnishi et al. 2011, 2012)

 Differentiation of the four genera
 Identification of Megasphaera species
 27/09/2012 22

Grouping of VTT Culture Collection

Pectinatus strains in ribotyping with EcoRI enzyme

Suihko & Haikara 2001, Juvonen & Suihko 2006

 27/09/2012 23

Eradication of contamination

 Regular hygiene control and rapid counter-measures

 detection in the filling area is always a risk

 Finding of contamination sources important

 ”all” containers -> go through whole process
 sporadic containers -> focus on filling
 anaerobic (micro)niches, brewing environment

 Intense mechanical cleaning and disinfection

 Rather sensitive to heat treatments (10-15 PU) and

common biocides (peracetic acid effective)
 biofilms 10-100 more resistant to biocides
 thermal adaptation possible
 27/09/2012 24

Conclusions
Biodiversity and ecology

 The group of strictly anaerobic beer-spoilage bacteria

constitutes six species
 new: P. haikarae, M. sueciensis, M. paucivorans

 P. portalensis, P. brassicae and P. sottaceto potential spoilers

linked with the production of wine and pickled vegetables

 Possible habitats of Pectinatus bacteria widened from the beer

brewing to other fermentation processes

 Beer-spoilage Pectinatus spp. and M. cerevisiae appear to be

uniquely adapted to the brewing environment
 Selenomonas, Zymophilus and new Megasphaera possibly
occasional invaders with plant material or water
 27/09/2012 25

Conclusions
Contamination and its control

 Contaminations possible everywhere in the brewing process

 More stress resistant than previously thought

 The routine cultivation methods also detect the new spoilers

 The EnBase System is a promising new medium for accelerated
detection of Pectinatus bacteria in beer
 DNA based methods most accurate and relaibale for identification
and tracking of contaminations
 many methods already developed for the new spoilage species
 Maintenance of good hygiene and rapid counte-measures are the
key control methods
 27/09/2012 26

Conclusions
Future
 ’

 Growing importance as spoilage organisms due to increased

production of beverages with reduced antimicrobial hurdles
 Growth potential in novel drinks now shown

 A beneficial role in biohydrogen production from organic waste

and possibly also in anaerobic wastewater treatment

 Better understanding of global distribution and behaviour

(including survival strategies) is required for developing more
effective control strategies
 27/09/2012 27

Acknowledgements

 VTT Technical Research Centre of Finland

 Dr Erna Storgårds and Dr Outi Priha
 MSc Vertti Virkajärvi

 PBL Brewing Laboratory

 owned by Polttimo Oy, Viking Malt Reso Oy, Oy Hartwall Ab,
Oy Sinebrychoff Ab and Olvi Oyj

 BioSilta Oy, Finland

 27/09/2012 28

VTT - 70 years of
technology for business
and society
 27/09/2012 29

Where are the ”old species” found?

 Isolated only from beer and/or breweries

 Pectinatus worldwide, M. cerevisiae more restricted

 most isolates from spoiled beers

 common in filling halls
 occassionally in fermentation areas

 Zymophilus spp. and S. lacticifex

 Germany, Finland
 pitching yeast, brewery waste
 27/09/2012 30

Identification – the traditional approach

Discrimination of beer-spoilage Pectinatus

Characteristic P. cerevisiiphilus P. frisingensis P. haikarae

Catalase activity – – +
Growth at 37oC + + –
Acid production from:
D-cellobiose – + –
i-inositol – + +
Lactose – – +
-D-melibiose + – +
N-acetyl-glucosamine – + –
D-salicin + + –
D-xylose + – +
+, growth/activity, - no growth/activity
 27/09/2012 31

Identification – the traditional approach

Discrimination of beer-spoilage Megasphaera

Characteristic M. cerevisiae M. paucivorans M. sueciensis

Cell size (μm) 1.5–2.1 1.2–1.9 x 1.0–1.4 1.0–1.4 x 0.8–1.2

Growth rate (PYF medium) 1–2 d 3d 4d

Organic acids from fructose + – –
Growth with lactate + – –

Major volatile fatty acids 1 C, iV, B iV, C iV, B, C, V

1Relative amount is ≥ 10%. The products in bold-face constitute 40–60% of the total amount. B, butyric acid; iV,
isovaleric acid; V, valeric acid; C, caproic acid.
Symbols: +, positive; –, negative.
 27/09/2012 32

RFLP analysis of PCR products amplified with the An-0279f

and An-0603r primers and digested with KpnI, XmnI and BssHII

Zymophilus
Selenomonas
Megasphaera
Pectinatus

Juvonen et al. 2008, Int. J. Food Microbiol. 125:162-169.

 27/09/2012 33

Minimum pH values and maximum

ethanol levels for growth in beer
pH

Zymophilus
5.0 • Z.paucivorans
•Z. raffinosivorans

4.5 Megasphaera/
Selenomonas
• M. cerevisiae,
•S. lacticifex Pectinatus
• P. frisingensis
• P. cerevisiiphilus
4.0
2 4 6
Ethanol, % (w/v)
 27/09/2012 34

Conclusions
 Growing importance anticipated due to the development of
increasingly sensitive low-alcohol beers and soft drinks

 P. haikarae, M. sueciensis, M. paucivorans new strictly anaerobic

beer-spoilage species

 P. sottaceto and P. portalensis are newly discovered plant-related

species able to cause beer spoilage if they find their way to breweries

 The ”old” species appear as permanent inhabitants in breweries

 Natural reservoirs of beer-spoilage Pectinatus bacteria and M.

cerevisiae remain an open question
 Contaminations possible everywhere in the brewing process

 Good stress tolerance can partly explain the persistence of P.

frisingensis and M. cerevisiae in breweries
 27/09/2012 35

Conclusions

 P. frisingensis, P. cerevisiiphilus and M. cerevisiae highly adapted to

beer brewing conditions (association with LAB and yeasts)
 The new Megasphaera species possibly occasional invaders in
breweries, mainly inhabiting anaerobic fermentation of plant material.
 P. portalensis, P. brassicae, P. sottaceto associated with lactic acid
and ethanol fermentations of plant-based foods and beverages
 Pectinatus and Megasphaera are potential hydrogen producers from
organic waste through acid fermentation and may participate in
anaerobic wastewater treatment processes.
 More information needed for understanding global distribution and
behaviour in environment
 27/09/2012 36

FORMATION OF SULPHUR COMPOUNDS (mg/l) IN CULTURE

MEDIUM AND IN BEER NATURALLY CONTAMINATED WITH
PECTINATUS
Culture Contaminated Taste
medium beer threshold
Methyl 14 - 80 20 1-3
mercaptane
Dimethyl 1.3 - 13.0 0.4 5 - 20
disulphide
Dimethyltri- 0.5 - 2.8 0.1 0.1
disulphide
Hydrogen 20 - 290 300 5 - 30
sulphide
VOLUME OF HEADSPACE AIR IN BOTTLED BEER
27/09/2012 37

1960-1990