center for biofilm engineering - amazon s3...draught line field samples jesse hiott, owner of prime...

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

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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