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Darla Goeres, PhD
Associate Research Professor
Center for Biofilm Engineering, MSU
Jesse Miller, PhD
Director of Applied Research
NSF International
Center for Biofilm Engineering
Draught Quality Summit | September 2018
Kelli Buckingham-Meyer, SBML
Biofilm bacteria are a self-organized, cooperative
community of microorganisms embedded in a
matrix of extracellular polymeric substances.
Head: Cambria bold, 32-40 pt, centered
Artist: P. Dirckx
Artist: P. Dirckx
Artist: P. Dirckx
Artist: P. Dirckx
SBML
Artist: P. DirckxL. Lorenz, SBML
Artist: P. Dirckx
L. Lorenz, SBML
The Medical Arena
Surfaces Around The Home
wjonespainting.com acrylicbath.com
tshirts.com
Broadwaycarpet.co.uk
?
Toothbrush
?
Nuk
?
Showerhead
?
Sink Strainer
?
Sponge
Look Familiar?
Its an Ecosystem!
Food Processing Workflow
csmonitor.com
zehabesha.com
What is in the Literature?
zehabesha.comcsmonitor.com
Tolerant to disinfectants & antibiotics
Public health
Structure & equipment degradation
Safety
Aesthetics & taste
Bioremediation & biofuels
Why do we about care biofilm?
www.secolo.co.za/events/microbial-influenced-corrosion-mic-workshop/
Efficacy test method determines the
log reduction of viable cells
Removal method determines the
elimination of the biofilm matrix, which
includes more than just viable cells
Efficacy vs. Removal
Efficacy vs. Removal
KILL REMOVAL=
Great Tasting Beer
Brewer
Consumer
Tavern
Owner
Distributor
What are the stakeholders needs & concerns?
Design a reproducible biofilm that can
be used for efficacy studies by
manufacturers and servicers of beer
line cleaning chemistries.
Measurement of biofilm reduction will
allow the BA to assess cleaning
efficiency and protect the
manufacturers of craft beer.
Goal
Long Draw Draught System
Long Draw Draught System: Clean-In-Place
Draught Line Field Samples
Jesse Hiott, owner of Prime Lines, sent the CBE 11
draught beer lines that were removed from a keg box.
The beer type was recorded as “all.”
The lines had been in place for 3.5 years and beer stones
were visible.
It had been 11 days since the lines were last cleaned.
CBE imaged and sampled the lines for viable bacteria and
yeast.
Results: Viable Bacteria and Yeast Counts
Bacteria Counts Yeast Counts
Log10(CFU/cm2) Log10(CFU/cm2)
1 <1 <1
2 <1 1
3 4.4 4.5
Sample
Results: Line 1 Images
Tubing wall integrity is
compromised, unidentified
substances collect in holes.
L. Lorenz, 25X L. Lorenz, 25X
Limited number of cell
clumps found, supports
viable count data.
Results: Line 1 Images
Tubing wall integrity is
compromised, unidentified
substances collect in holes.
L. Lorenz, 25X L. Lorenz, 25X
Limited number of cell
clumps found, supports
viable count data.
Results: Line 2 Images
Holes are not consistent
across the tubing surface.
L. Lorenz, 25X L. Lorenz, 25X
Accumulation of undefined
debris is apparent.
Results: Line 3 Images
Line 3 contained more biofilm
clusters than lines 1 & 2.
L. Lorenz, 25X L. Lorenz, 63X
Zoomed in image of a biofilm
cluster containing large and small
cocci. Images support viable count
data.
Full Scale Experiments
Schematic Overview
Dispense line & faucet
Keg coupler
Beer manifolds
Gas manifolds
CO2 tank & regulator
Dispense line coupler
Kegerator
Credit: Brewers Association & New Belgium Brewing
Beer Lines: 3 Tubing Types
Credit: Brewers Association & New Belgium Brewing
Coupler/Simulated Keg
5/16” vinyl
tubing (5 ft.)
5/16” PET
tubing (15 ft.)
3/16” vinyl
tubing (3 ft.)
Faucet
A B C
Shank and faucet
Pre-conditioning the Draught System
Cleaning canister used to
pre-condition lines with
3% NaOH.
Lines were rinsed with
tap water, then beer.
Draught System: Inoculation
Draught System: Inoculation
Inoculated at Time 0, 6 &
12 weeks.
Poured beer intermittently
and sampled tubing
(closest to the tap) and
beer weekly.
Pouring Beer
Draught System: Sampling
Draught System: Sampling
Bacteria enumerated on Universal Beer Agar with
cycloheximide.
Yeast enumerated on WL Nutrient Agar.
Draught System: Bacteria & Yeast LD Repeatable
20151050
4
3
2
1
0
Time since last inoculation
Ba
cte
ria
LD
1
2
3
4
Faucet
Bact
eria L
og
10
CFU
/cm
2
20151050
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
Time since last inoculation
Ye
ast
LD
1
2
3
4
Faucet
Yeast
Log
10
CFU
/cm
2
Bacteria and Yeast Grow Together in Biofilm
543210
4
3
2
1
0
Yeast LD
Ba
cte
ria
LD
A
B
Location
Biofilm
Bacteria in biofilm undergoes exponential decay
121211714108721
4
3
2
1
0
Days since Inoc.
Ba
cte
ria
LD
4/6/2017
5/15/2017
8/1/2017
Inoc. Date
C tubing: biofilm
Yeast counts also decay exponentially
121211714108721
4
3
2
1
0
Days since Inoc.
Ye
ast
LD
4/6/2017
5/15/2017
8/1/2017
Inoc. Date
C tubing: biofilm
Day 125 Results: Faucet 2
Tubing A Tubing B Tubing C
Day 125 Results: Faucet 3
Tubing A Tubing B Tubing C
Draught System: Flow Characteristics
In draught systems, flow is
intermittent. High fluid shear is
introduced each time a beer is
poured, which in the short term, could
result in removal of biofilm.
3% NaOH efficacy experiment
Grow Pseudomonas aeruginosa
biofilm in beer tubing for 24 hours
at room temperature.
At 24 hours, sample tubing for
biofilm.
Pump 3% NaOH for 15 minutes
through tubing.
Rinse with for 10 minutes with
sterile tap water.
Sample tubing for biofilm.
3% NaOH treatment results in a 4.4 log reduction of Psd. aeruginosa biofilm
SampleMean
Log10(CFU/cm2)
Standard
Deviation
Log
Reduction
(pre – post)
Pre-Treatment 6.0 0.16
4.4Post-Treatment 1.6 0.49
3% NaOH treatment results in removal of Psd. aeruginosa biofilm
Pre-treatment:
Post-treatment:
250x 250x
250x250x
630x 630x
630x 630x
Psd. aeruginosa biofilm has a similar appearance to biofilm that consists of yeast and bacteria, although bacterial density is about double (6 Logs vs 3 Logs)
Psd. aeruginosa biofilm Yeast + bacteria biofilm
Draught System: Challenges
Foaming/Temperature
CO2 pressure
Slow growth of bacteria
Bacterial numbers decay over time
Sampling is destructive
Going back to the simple system
Artificially age clear vinyl beer
draught line tubing (3/16 inch).
Grow suspended cultures of A.
aceti, P. damnosus, L. rhamnosus
and S. cerevisiae.
Add cultures and one beer to flask
kept at room temperature.
Recycle beer and bugs for 1 hour,
then let sit for 24 hours. Repeat.
Remove foam & bubbles using
bubble trap.
Analyze biofilm accumulation.
Going back to the simple system: results
L. Lorenz, 25X L. Lorenz, 63X
24 hour old biofilm 48 hour old biofilm
Mean Bacteria Biofilm Density = 5.6 +/- 0.07 Log10(CFU/cm2)
Mean Yeast Biofilm Density = 5.2 +/- 0.44 Log10(CFU/cm2)
Path Forward
Efficacy will be done at NSF International
Unanswered questions:
What is a “typical” mature beer biofilm?
When does biofilm start to affect beer taste/smell?
Acknowledgements
Brewers Association Chuck Skypeck
Neil Witte
Damon Scott
New Belgium Brewing Company Matt Meadows
Josh Van Zee
Matt Simpson
Cardinal Distributing Doug “the Beerbarian” Bailey
Scott Pitcher
Cash
Bronken’s Distributing Bruce
George Taylor
NSF International Jesse Miller
Bryan Schindler
Zachery Guerin
Atwain Atwain
Christine Greene
Sierra Nevada Brewing Company
Standardized Biofilm Methods
Laboratory Team Darla Goeres
Al Parker
Lindsey Lorenz
Kelli Buckingham-Meyer