Monitoring Fermentation With an Aber Yeast Monitor

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Research & Development 1/30 Monitoring fermentation with Aber yeast monitorOptimisation of Products & Processes

T\PSData\ArchivePS\paee\General\Instrumentation\\Instructions-applications\quality\Yeast conc\YCMMW002 Monitoring fermentation

with Aber yeast monitor.doc

Monitoring fermentation with

Aber Yeast Monitor

Heineken Technical Services B.V.

Research & Development

Optimisation of Products and Processes

Zoeterwoude, 27 November 1998

Type of Report : Final

Principal : Zoeterwoude

Project Name : Yeast Concentration during fermentation

Author : M.M. de Wit

Project Manager : M.M. de Wit

Keywords : instrumentation, fermentation, yeast growth


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CONTENTS

1 SUMMARY ...................................................................................................................................... 4

1.1 Opportunity ........................................................................................................................... 41.2 Objective ............................................................................................................................... 4

1.3 Test equipment ...................................................................................................................... 4

1.4 Results ................................................................................................................................... 4

1.5 Conclusion............................................................................................................................. 5

1.6 Future developments ............................................................................................................. 5

2 INTRODUCTION ............................................................................................................................. 6

3 TEST EQUIPMENT ......................................................................................................................... 7

3.1 Aber Yeast Monitor 320 ........................................................................................................ 7

3.2 Coulter Counter ..................................................................................................................... 7

4 TEST ................................................................................................................................................. 8

4.1 Test objective ........................................................................................................................ 8

4.2 Test set-up ............................................................................................................................. 8

4.3 Trending parameter settings during test ................................................................................ 9

4.4 Test description / results........................................................................................................ 9

4.4.1 Phase 1: ................................................................................................................... 9

4.4.2 Phase 2: ................................................................................................................. 12

4.4.2.1 Process information: ............................................................................... 13

4.4.3 Phase 3: ................................................................................................................. 13

4.5 Summary results Yeast Monitor .......................................................................................... 13

5 CONCLUSIONS ............................................................................................................................. 15

ADDENDUM A ....................................................................................................................................... 16

Graph 1 .......................................................................................................................................... 17

Graph 2 .......................................................................................................................................... 18

Graph 3 .......................................................................................................................................... 19

Graph 4 .......................................................................................................................................... 20

Graph 5 .......................................................................................................................................... 21

Graph 6 .......................................................................................................................................... 22

Graph 7 .......................................................................................................................................... 23

Graph 8 .......................................................................................................................................... 24

ADDENDUM B ....................................................................................................................................... 25

B.1 How the Yeast Monitor 320 Works ................................................................................. 25

B.2 What and How the Yeast Monitor Actually Measures.................................................. 25

B.3 The Probe Constant .......................................................................................................... 26

B.4 The electrical properties of living systems...................................................................... 26

B.4.1 The relationship between capacitance and biomass...................................................... 26

B.4.2 Conductivity....................................................................................................................... 28


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B.4.2.1Maximum Conductivity Limit......................................................................................... 28

B.5 Other Influences on the Capacitance Measurement ...................................................... 29

B.5.1 Crosstalk ............................................................................................................................ 29

B.5.2 Electrode polarisation ....................................................................................................... 29

B.5.3 Gas Bubbles ....................................................................................................................... 29B.5.4 Temperature ...................................................................................................................... 30


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1 SUMMARY

One of the objectives of Heineken is to produce beer at a constant and high quality all over the world.

However, a recent investigation (October 1996) revealed that 50% of the lager in tanks and the final

product in the bottle (Heineken and Amstel) did not match all product specifications. (apcwk003.ip andapcwk004.ip)

Fermentation is a very important step in the brewing process because during this step many flavour

components are formed. The yeast is the determining factor for all the flavour components formed, and

is thus responsible for the ultimate quality of the fermented wort.

For this trial, an Aber Yeast Monitor (AYM) was installed that had four measuring probes. Three of the

probes were installed in the Apollo FV at different levels and one was installed in the filling line.

1.1 Opportunity

1. An in-line yeast concentration measurement system, that performs well, makes it possible to detectdeviations in yeast growth and hence the fermentation.

2. A yeast concentration signal could be an input for an adaptive fermentation control system (seereport: apcwk001.rep). Then, it would be possible to correct deviations in fermentation that effect

product quality.

1.2 Objective

The Aber Yeast Monitor was tested to determine whether it could measure yeast concentration to within

2 million yeast cells per ml. This is comparable to the Coulter Counter.

1.3 Test equipment

The AYM, type 320, consists of a sensor, pre-amplifier and electronics. Specifically for this trial, it was

equipped with a multiplexer to allow 4 probes to be installed on 1 electronic unit. The scan time foreach probe was 2 minutes. All the trending data was stored in the Foxboro AI control system.

As reference measurement for determining the cell amount and the cell size, a laboratory Coulter

Counter measuring device was used.

1.4 Results

1. The steepness of the measuring slope from the Yeast Monitor does not corresponding with the cellamount. (See Addendum A graph 1)

2. No relationship was found between cell diameter / cell volume and the steepness of the measuredoutput. (See Addendum A graph 2)

3. The maximum amount of yeast cells, measured by cell count, is achieved earlier than by the AYM.

4. In all the fermentations, probe 3 (5.6 metre from bottom) indicated a higher signal than probe 2 (9.8metre from bottom), whereas the sample measurements indicate the same amount of yeast cells.

5. The deviation between measuring probe 2 and 3 is not constant within the batch or over the batches.


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6. The deviation of the measuring probes and the cell count (Coulter Counter) is not constant within thebatch or over the batches.

7. The mounting position of the measuring probe is critical for measurement. Mounting the probe at anupwards-facing angle may cause problems i.e. sedimentation of yeast cells on the probe, resulting in

a constant high output signal.

8. The signals of the AYM were unreliable within the batches as well over the batches. Possible causesfor this problem are:

Temperature influence (day/night) on the probe electronics,

Variation of the yeast cell dimensions / yeast mass during the fermentation and themeasured value capacitance are not correctly related.

1.5 Conclusion

The Aber Yeast Monitor 320 is, in its current design, unsuitable for measurement of yeast

concentration during fermentation within 2 million cells/ml.

1.6 Future developments

The supplier will do further research on yeast cell behaviour related to the measuring principle and the

influence of temperature on the electronics. When they are successful in optimising the AYM they will

inform Heineken.


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2 INTRODUCTION

One of the objectives of Heineken is to produce beer at a constant and high quality all over the world.

To fulfil this objective a lot of research has taken place to ensure a constant quality of the product in all

the process steps. Despite all this effort an investigation in October 1996 revealed that about 50% of thelager tanks and the final product in the bottles of Heineken and Amstel did not match all product

specifications. (apcwk003.ip and apcwk004.ip)

As part of the project Control of Batch Fermentations (see report: apcwk001.rep) the AYM type 320

has been investigated. The AYM 320 is a further development of the AYM 316, used at the brewery

Zoeterwoude for yeast pitching. The distinction between the two models is the capability of the 320 to

measure lower yeast concentrations.

The behaviour of the yeast during the fermentation is a key parameter for the ultimate quality of the

product. Therefore the AYM was installed to see or it was capable to measure the yeast concentration

during fermentation.

During the fermentation it is of interest to know:

the amount of yeast cells to start the fermentation with,

the concentration of the yeast cells on different levels in the fermenter.

the speed of multiplication of the yeast,

the maximum amount of yeast cells,

the sedimentation speed of the yeast and

the resulting amount of yeast in suspension.


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3 TEST EQUIPMENT

The instrument to be tested was the AYM 320 of Aber Instruments LTD. To verify the performance of

the AYM, samples taken during the fermentation were analysed with a Coulter Counter and DMA.

3.1 Aber Yeast Monitor 320

The tested AYM 320 consists of;

electronic panel

4 channel multiplexer

electronic module for each probe and

4 measuring probes.The measuring probes are attached to the electronic modules with a connector. The electronic modules are

linked with the multiplexer by a special cable. The panel is provided with an indicator to monitor the

measured value of living yeast cell concentration. Data entry into memory for calibration parameters for up

to 5 individual strains is provided and these may be selected from the front panel.

There is provision for applying, automatically or manually, to the probe a high intensity cleaning pulse,

which removes solids or gas bubbles from the probe, which may interfere with the signal. Visual

indication of a number of potential fault conditions is also provided.

To facilitate integration of the meter into

automatic control systems, yeast concentration

and conductance readings are provided as 4 -

20 mA and 0 - 6 V outputs, respectively.

Similarly, fault conditions may be used to

trigger external alarms or other corrective

actions via relay switches. In addition, remote

configuration for individual strains is possible.

The probe consists of four platinum pinsinserted into a flat-ended plastic tube housing.

The pins transmit a high frequency radio

signal directly into a yeast slurry. This induces

a capacitance (i.e. charge difference between

the cell contents and the surrounding medium)

only in viable cells where the cell membrane

is intact. The probe can detect this and the

magnitude of the capacitance detected is then proportional to the viable biomass concentration in contact

with the probe. The probe also measures the conductance of the background medium.

3.2 Coulter Counter

The Coulter Counter model ZM is a laboratory instrument. It provides a particle size analysis over a

range 1 m to 30 m. It gives the number of particles within, or larger than, a pre-selected volume or

diameter size range contained in a known selectable volume of suspension.

The HTS laboratory experience for the total inaccuracy of sample handling and Coulter Counter is 2

million yeast cells per ml.

Figure 1: Probe with pre-amplifier.


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4 TEST

The equipment was installed on Apollo 372 (4.000 hl) at the brewery Zoeterwoude. In the period May

1997 till January 1998 the test was executed in 3 phases:

Phase 1: research of the correlation between measured value of the AYM and the Cell Count(Coulter Counter)

Phase 2: research of the correlation between measured value of the AYM and the Yeast cellvolume (Coulter Counter)

Phase 3: behaviour equipment without intensive attention.

4.1 Test objective

The AYM was tested to determine whether the indicated values during process fermentations were

within 2 million yeast cells per ml, compared with the Coulter Counter.

4.2 Test set-up

After set-up and calibration of the AYM in the laboratory the instrument was installed on the fermenter.

The four measuring probes were installed at the following positions (see figure 2):

probe 1 at the top of the cone, 3.4 meter from the bottom of the tank,

probe 2 in the cylindrical part, 5.6 meter from the bottom,

probe 3 in the cylindrical part, 9.8 meter from the bottom and

probe 4 in the filling line.

Figure 2: Test set-up, installed position of probes.

5

6

1. Y.M. probe 1

2. Y.M probe 23. Y.M probe 3

4. Y.M probe 4

5. Filling line

6. CO2 outlet

5.6

m

3.4m

9.8m

3

2

1

4

Aber Yeast Monitor

Multi plexer

Fox. control system

(data trending)


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4.3 Trending parameter settings during test

1. Aber Yeast MonitorThe AYM was provided with a 4-channel multiplexer. This option offered the measuring device to

handle 4 input sensors. During all tests the sensor inputs were measured sequentially, with a 2minutes time interval.

2. Foxboro DCS systemThe output signal of the AYM and the temperature of the fermentation vessel were continuously

trended in the DCS (Distributed Control System) system. For processing the graphs the average

measuring value during 1 hour was used.

4.4 Test description / results

To verify the recordings of the AYM, samples were taken from the process and analysed in the

laboratory.

The extract value was determined with the DMA and with the Coulter Counter the amount of yeast cells

and yeast cell diameter distribution was determined. The dry weight determination was executed inaccordance with a standard laboratory procedure (certain amount of sample liquid, filter sheet, drying in

an oven and weighing).

4.4.1 Phase 1:

Description:

This part of the research deals with the correlation between measured value of the AYM and the Cell

Count values (Coulter Counter) of the samples.

The sampling frequency for these 10 fermentations was:

2 samples every 24 hour for extract, dry weight and yeast cell measurement, sample point level 2 (near mounting position probe 2), fermentation 2 till 11.

Results:

(Graphs of the individual batches are reported in appendix report YCMMW003.rep.)

In the first batches, in spite of optimisation of the measuring equipment, a mismatch between the AYM

and the analysed sampling values remained.

In consultation and with co-operation from the supplier a re-calibration with the probe simulator and a

software up-date from version 1.0 to 1.2 took place after the 4th

fermentation. In the time period

between the 5th

and 11th

fermentation regular checks and manual cleaning of the probes were executed.

None of the executed maintenance and optimisations has resulted in a better performance of the AYM.

Probe 3 indicated in all recorded batches a significant higher value than probe 2. The deviation between

probe 2 and 3 was not constant within the batch as well over the batches. The measuring data of probe 2as well probe 3 deviate from the sample values.

Probe 1, installed in the cone of the fermenter under a certain angle, indicated only during filling up

relevant data, soon after filling the maximum output signal (>20 mA) was reached. This not correct

functioning of probe 1 was probably caused by yeast sedimentation on the probe. Probe 4 indicated the

yeast content during filling up, during the fermentation the output signal was constantly over-saturated

due to yeast sedimentation in the pipe wherein the probe was mounted.


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The results of 3 fermentations, with an identical temperature profile, are plotted in the graphs below.

In the first plot we see the 3 Es curves and the average temperature profile of the 3 fermentations. The

second plot shows the cell count determined by the analysed samples. Plot 3 indicates the difference

between the measured values of the AYM minus the sample values, while plot 4 shows the registered

values of the AYM.

The following conclusions of these plots can be noted:

The steepness of the cell count measurement deviate to the measurement of the AYM, The top of the cell count measurement is achieved after 120 hours while the top by the

AYM is registered approx. 20 hours later, The graph indicating the differences between the AYM and the cell count shows

deviations within the batch as well over the batches of 5 million cells/ml,

The desired accuracy of 2 million cells/ml is not met.

3 Amstel fermentations

Ferm. 3 Ferm. 4 Ferm. 7 Temp.

Cell count Coulter Counter

0

10

20

30

40

50

60

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Time in hours

Yeastcells/ml(x1million)

Cell registration YM2

0

10

20

30

40

50

60

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Time in hours


Es

0

2

4

6

8

10

12

14

16

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Time in hours

Es(degr.Plato)orTemp.(degr.C)

Difference YM2 - Cell Count

-15

-10

-5

0

5

10

15

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Time in hours



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In the 9th

fermentation, fermented at a higher

temperature, the same profiles can be

observed. An even stronger deviation in

steepness of the yeast growth and a higher

difference in cell measurement wereregistered. Due to the higher fermentation

temperature the top of maximum yeast cells

concentration is reached after 100 hours.

Also in this fermentation, as all the other

measured batches, the AYM indicated the top

at a later time.

Conclusions of the 9th

fermentation:

The steepness of the cell count

measurement deviate to themeasurement of the AYM,

The maximum amount of yeast cellsmeasured by cell count is achieved

earlier then by the AYM,

The graph indicating the differencesbetween the AYM and the cell count

shows deviations of 10 million

cells/ml,

The desired accuracy of 2 millioncells/ml is not met.

In consultation with the supplier the possible causes for the mismatches were analysed. As most

possible causes were pointed out:

The change of the yeast cell diameter, while the AYM measures the capacity of the yeast

cells, the change in diameter thus the change in total volume might change the measuredsignal.

Disturbances of the environment on the measuring probes. (To eliminate environmentaldisturbances the calibration of the probes has to be executed on the vessel.)

Another reason to continue with phase 2 was to research the homogeneity of the fermentation vessel.

The AYM gave continuously a higher yeast concentration on level 3 than on level 2 and that is in

contradiction with the expectations of the yeast specialists.

Cell count Coulter Counter9th fermentation

0

10

20

30

40

50

60

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Time in hours


cell-count

Amstel fermentation

Cell registration YM2 and YM 39th fermentation

0

10

20

30

40

50

60

70

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Time in hours


YM3-top

YM2-roof

Differences cell measurement9th fermentation

-30

-20

-10

0

10

20

30

40

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Time in hours


YM3-YM2

YM2-cell cnt level 2

YM3-cell cnt level 2


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4.4.2 Phase 2:

Description:

During 2 fermentations not only the number of yeast cells was determined but also the distribution of

yeast cell diameters and total yeast volume. Besides the higher sample frequency, the samples weretaken on 2 different levels. The onsite calibration of the measuring probes was carried out before the

13th

fermentation.

The sampling frequency for these 2 fermentations was as follows:

3 till 6 samples every 24 hour for extract, dry weight and yeast cell measurement, sample point level 2 and 3 (near mounting position probe 2 and 3), fermentation 12 and 13.

Results:

(Graphs of the individual batches are reported in an appendix report)

By filling up the vessel with 2 batches of wort (without yeast) 3 of the 4 probes were covered by the

liquid. It was not possible to calibrate probe 3, mounted at the highest point in the vessel, under processconditions. Probe 3 was therefore calibrated with a special metal beaker (special made by Aber) to

simulate the process conditions. As we can see in the graphs (addendum A graph 1, 2 & 3) the

calibration has not improved the performance of the AYM.

During the 12th

and 13th

fermentation samples were taken at level 2 (5.6m) and level 3 (9.8m). With the

Coulter Counter the average cell size of the yeast was determined and the total cell volume calculated.

In graph 1 of addendum A, the sample value of the cell count is plotted from level 2 and 3. According to

the sample values a quite homogeneous yeast concentration is obtained till 25 million cells per ml.

Above the 25 million cells the yeast cell concentration on level 2 is slightly higher.

In graph 2, the recorded values are plotted against the total cell volume. This graph shows that there is

no difference between the mismatch of the AYM related to the cell size or cell volume.

No improvement is realised between the concentration differences of YM2, YM3 and the sample values

(see graph 3).Resulted the following conclusion can be noted:

Homogeneous yeast cell concentration up till 25 million cells per ml, The steepness of the yeast growth measured by the AYM and the sampling measurement,

related to total yeast cell volume, is not identical,

There is still a considerable deviation between probe 2 and 3, The deviation between probe 2 and 3 is not constant during the fermentation, The graph indicating the differences between the AYM and the cell count shows

deviations of 5 million cells/ml.

The data were discussed with Aber Instruments LTD, but no concrete causes could be assigned for the

mismatch. In an open consultation with the supplier possible causes were discussed, like: Temperature influence (day / night) on the probe electronics,

Variations of the yeast cell dimensions in combination with yeast cell mass during thefermentation.

The supplier will do further research on yeast cell behaviour related to the measuring principle and the

influence of temperature on the electronics. When they are successful in optimising the AYM they will

inform Heineken.


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4.4.2.1Process information:Many data gained during phase 2 has interesting information on the process. In the graphs 4 till 8

(addendum A) this data is plotted. In consultation with the yeast specialists within Heineken the main

conclusions of these graphs are noted below:

1. Graph 4: The average cell volume in this graph shows peaks at 24, 48, 65 and 84 hours. Those peaks are

probably related with the cell division.

2. Graph 5: For the first 96 hours there is a correlation between cell volume and cell mass, after 96 hours the

cell mass deviates from the cell volume most likely caused by the take up of glucose by the yeast

cell resulting in a higher cell mass.

3. Graph 6:

There is a good relation between cell size and cell volume.

4. Graph 7: The dry weight samples for point 2 and 3 are similar.

5. Graph 8: The cell count samples for point 2 and 3 are similar up till 25 million cells. Thereafter, they

deviate

4.4.3 Phase 3:

Description:

The last 4 fermentations were recorded to see the behaviour of the equipment without intensiveattention.

The sampling frequency for the last 4 fermentations was as follows:

1 sample every 24 hour onlyfor extract measurement, sample point level 2, fermentation 14, 15, 16 and 18.

Results:

(Graphs of the individual batches are reported in an appendix report)

The trended AYM information of 2 batches was disturbed in such a way that the information was not

usable. A cause for the disturbed information was not found, so we can conclude that the Yeast Monitor

needs constant attention to functioning properly.

4.5 Summary results Yeast Monitor

Measuring probe 1:

Measuring probe 1, mounted in the cone of the fermenter, did not function correctly. At the start of the

fermentation the probe produced measurements within the range of the instrument, but soon after the


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output signal is over saturated. This incorrect functioning of the probe is probably caused by the

mounting position. In the cone the measuring probe measures under a certain angle, which causes

sedimentation of yeast between the measuring pins of the probe.

Measuring probe 2:Measuring probe 2, mounted 5.6 m from the bottom of the tank, did function during all fermentations,

but the deviations with the sample values were not consistent within the batches and not reproducible

over the batches (see graph 4). Besides the steepness of the measuring signal was not identical to the

curve of the sample signal (see graph 1 and 2).

Measuring probe 3:

Measuring probe 3, mounted 9.8 m from the bottom of the tank, did function during all fermentations.

The indication was in all fermentation higher than the measured value of probe 2 (see graph 1).

Measuring probe 2 and 3:

The difference between the probe 2 and 3 was fluctuating within the batches (see graph 3) as well overthe batches.

Measuring probe 4:

This probe mounted in the filling line indicated the yeast content during filling of the fermenter. During

the fermentation the output signal was constantly over saturated due to sedimentation of the yeast.

Electronics

During all tests there was no drop out of electronic components, but in 4 batches the recorded signal

was so noisy / disturbed that it was useless. For the batches 11 and 12 the cause was found in an active

cleaning pulse of the probes generated by the main electronic. After switching off this feature the

disturbance disappeared. For the disturbances recorded in the batches 15 and 18 no explanation was

found.


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5 CONCLUSIONS

1. The steepness of the measuring slope from the Yeast Monitor is not corresponding with the cellamount. (See Addendum A graph 1)

2. No relation has been found versus the cell diameter / cell volume and the steepness of the measuredoutput. (See Addendum A graph 2)

3. The maximum amount of yeast cells measured by cell count is achieved earlier then by the AYM.

4. In all the fermentations probe 3 (5.6 meter from bottom) indicated a higher signal than probe 2 (9.8meter from bottom), whereas the sample measurements indicate the same amount of yeast cells.

5. The deviation between measuring probe 2 and 3 is not constant within the batch or over the batches.

6. The deviation of the measuring probes and the cell count (Coulter Counter) is not constant within thebatch or over the batches.

7. The mounting position of the measuring probe is critical for the measurement. Mounting the probe inan angle facing up might cause problems by sedimentation of yeast cells on the probe, resulting in a

constant too high output signal.

8. The signals of the Y.M. were not reliable within the batches as well over the batches. Possible causesfor this disfunction is:

Temperature influence (day/night) on the probe electronics,

Variation of the yeast cell dimensions / yeast mass during the fermentation and the measuredvalue capacitance are not correctly related.

9. During the fermentation the yeast cell concentration was homogeneous up till 25 million cells/ml.

Over all conclusion:

The Aber Yeast Monitor 320 is, in its current design, not suitable to measure the yeast

concentration during fermentation within 2 million cells/ml.


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ADDENDUM A

Yeas

tgrowthAmstelferment

ation

13thfermentation

010

20

30

40

50

60

0

24

48

72

96

120

144

168

192

216

240

264

288

312

336

360

3

84

Timeinhours


0246810

12

14

16

18

EsorTemp.

YM3-top

YM2-roof

cellcntlevel2

cellcntlev

el3

Es

Temp.


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Graph 1


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Graph 2

Yeast

growthAmstelfermentation

13thfermentation

010

20

30

40

50

60

0

24

48

72

96

120

144

168

192

216

240

264

288

312

336

360

384

Timeinhours

Numberofyeastcells/ml(x1million)

Totalcellvolume(micron^3/200)

0246810

12

14

16

18

EsorTemp.

YM3-top

YM2-roof

Totalcellvol.

Es

Temp.


19/30




Graph 3

Differen

ceYeastindication:YM3

-YM2

13thfermentation

0246810

12

14

16

18

0

24

48

72

96

120

14

4

168

192

216

240

264

288

312

336

360

384

Timeinhours


YM3-YM2

Temp.


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Graph 4

Average:Cell

volume,DryWeightand

Cellcount

13thfermentation

0510

15

20

25

30

35

40

45

50

0

2

4

48

72

96

120

144

168

192

216

240

264

Timeinhours

Yeastcells(x1million)and

DryWeight(/10)

150

180

210

240

270

300

Cellvolume(microns^3)

Avg

.D.W.(x10)pnt2and3

A

vg.CellCnt.,pnt2and3

Avg.cellvol.microns^3pnt2and

3


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Graph 5

Avg.ce

llvolumeandDryWeight/cellcount

13thfermentation

0.0

9

0.1

0

0.1

1

0.1

2

0.1

3

0.1

40

2

4

48

72

96

120

144

168

192

216

240

264

Timeinhours

DryWeight/cellcount

150

180

210

240

270

300


Avg.cellmasssamplepnt2and3

Avg.cellvol.microns^3pnt2and3


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Graph 6

A

vg.cellsizeandcellvolume

13thfermentation

6.80

7.00

7.20

7.40

7.60

7.80

8.00

8.20

0

24

48

72

96

120

144

168

192

216

240

264

Timeinhours

Cellsize(microns)

150

180

210

240

270

300


Avg.cellsizemicrons,pnt2a

nd3

Avg.cellvol.m

icrons^3,pnt2and3


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Graph 7

Dryweightsamplepoint2and3

13thfermentation

012345

0

24

48

72

96

120

144

168

192

216

24

0

264

Timeinhours

Dryweight(g/l)

samplepnt2

sample

pnt3


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Graph 8

Cellc

ountsamplepoint2an

d3

13thfermentation

0510

15

20

25

30

35

40

0

24

48

72

96

120

144

168

192

216

24

0

264

Timeinhours

Cellcount(cell/ml)

s

amplepnt2

samplep

nt3


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ADDENDUM B

B.1 How the Yeast Monitor 320 Works

For routine use of the instrument, it is not necessary to understand its principle of operation in detail. Ashort introduction to the theory on which the instrument is based will, however be of interest and may

be of assistance in avoiding problems in application or interpretation of the results obtained from the

instrument.

B.2 What and How the Yeast Monitor Actually Measures

Any biological cells with intact plasma membranes in a suspension act like tiny capacitors when an

electric field is applied to that suspension. This is because a cell's plasma membrane is a poor conductor

and it separates the cell's conducting cytoplasmic material from the cell wall (if present) and the other

conducting material outside the cell, allowing a build up of charge around the plasma membrane.

In brewery applications we are primarily interested in the measurement of the quantity of viable yeast

being transferred into or out of the process. As yeast is a biological cell it exhibits this capacitance,

providing the cell membrane is intact. Only viable cells have intact membranes.

The Yeast Monitor 320's electrode probe has four platinum pins that make contact with the cell

suspension. A radio-frequency sine wave is applied to the outer two pins causing an electric field that

extends some 20 mm from the probe body. This affects all of the nearby cells with intact plasma

membranes; the ions inside and outside these cells move towards the plasma membranes, polarising

them.

The field being applied is accurately monitored by the two inner pins of the electrode probe and,

together with a current measurement, is used to determine the capacitance value.

The measured capacitance value is directly proportional to the amount of viable cells present.

Therefore, as the concentration of cells increases, so does the capacitance value. The system is

insensitive to non-viable cells (i.e. those with leaky membranes), gas bubbles and most non-biomass

solids in the suspension because none of these polarise significantly and therefore do not contribute a

significant capacitance.

The raw capacitance reading given by the Yeast Monitor 320 for a given slurry, will not only be due to

any cells present. The suspending medium (wort for example) also produces capacitance. This is usually

much lower than that of the cells. The probe itself will also add a constant background capacitance.

However the capacitance contribution from the probe and the medium can be easily isolated from thatof the cells by zeroing the instrument whilst the probe is immersed in cell-free medium.

The capacitance measured is actually proportional to the volume fraction of viable cells in the

suspension.


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The Yeast Monitor 320's display can be calibrated to provide measurement of viable yeast

concentrations in % viable volume or viable cells/ml. These factors must be determined by analysis in

the laboratory during commissioning and calibration.

B.3 The Probe Constant

The capacitance and conductance measured will be a function of the size and shape of the electrodes,

and any gain constants inherent in the measuring system. For generality of data, and easy comparisons

with data from other sources, it is common to work with "intensive" properties i.e. those that depend

only on the material being measured and are therefore independent of the measuring system ( electrode

size etc.). The intensive variables used in the Yeast Monitor 320 are :

Standardised Capacitance = K x measured Capacitance

Conductivity = K x measured Conductance

where K is the Effective Probe Constant. It depends on the probe's electrode geometry, the Head

Amplifier gain, and the Yeast Monitor 320's gain. It has the units of cm-1.

In normal measurement mode, the Yeast Monitor 320 will display Conductivity. Although standardised

capacitance is calculated in the software, it is always converted to % VV or cells/ml before being

displayed. The internal software does the multiplication by K.

The Effective Probe Constant, K, is found by multiplying together the constants for the probe, the

Head Amplifier, and the main instrument:

K = Kp * Kh* Km

The standard probe supplied with the Yeast Monitor 320 has a constant, Kp = 1.3

The value of K for the system, as supplied, is entered into the Yeast Monitor 320's memory at the

factory, and should not need to be changed unless a non-standard probe or Head Amplifier is fitted.

When the Instrument is switched to any Strain Position, the stored value of K is used to calculate

Conductivity (mS/cm), and Standardised Capacitance automatically. The resultant display of Viable

Yeast Concentration is in terms of % viable volume or cells / ml as selected by the Maximum

Concentration switch.

B.4 The electrical properties of living systems

B.4.1 The relationship between capacitance and biomass

The density (mass per unit membrane-enclosedvolume) of any particular type of biological cell does

not vary greatly with physiological status, therefore for a given cell type, Biomass will be directly

proportional to bio-volume.


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The Yeast Monitor 320 effectively measures bio-volume, i.e. the volume fraction P of the suspension

that is enclosed by the cytoplasmic membrane of the cells, and this is therefore directly proportional to

the Biomass Concentration.

This figure can be equated to viable yeast concentration.

The instrument will be more sensitive to large cells, and less sensitive to small cells such as bacteria.

An approximate guide to the capacitance readings expected, for a yeast concentration of 1 mg/ml dry

weight, is of the order 0.9 to 1.9 pF/cm. With the STRAIN switch in the TEST position, and a standard

probe, one unit on the display is equivalent to 2 pF/cm.

A more general guide is given in the graph of Figure 1

Figure 1 -- The Effect Of Cell Size On Yeast Monitor 320 Reading


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The Yeast Monitor 320 therefore provides the means

(i) to measure C at a pre-set frequency,

(ii) to correct this for the background with no yeast present, and(iii) to correct for the changes in yeast suspension capacitance caused by conductivity variation.

(iv) to output the data as % viable volume, or as cells/ml, after calibration against the method chosen

by the user.

(v) to correct for non-linearity's which occur at high yeast concentrations.

B.4.2 Conductivity

In addition to capacitance measurement, the Yeast Monitor 320 also provides a measure of the electrical

conductance, which is converted to Conductivity (mS/cm) using the stored value of K.

B.4.2.1 Maximum Conductivity Limit

For fundamental technical reasons, there is an upper limit to the conductivity at which it is

recommended to use the Yeast Monitor 320, beyond which values measured become unreliable.

Presently, with a standard probe and Head Amplifier, this limit is 7.5 mS/cm.


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B.5 Other Influences on the Capacitance Measurement

B.5.1 Crosstalk

Large changes in conductivity of the yeast suspension give rise to small artifactual capacitance changesdue to phase errors in the measuring system. This phenomenon is known as crosstalkand should not

normally be of sufficient magnitude to cause problems in brewery applications.

B.5.2 Electrode polarisation

A further conductivity-dependent effect, which occurs in practical measurements, is electrode

polarisation. This gives additional crosstalk such that the capacitance reading will increase slightly with

increasing conductivity. This increase is not stable, as it depends on the surface condition of the

electrodes, so it is difficult to correct for. However, the effect is only serious at very low frequencies, as

shown in fig 2 and so the Yeast Monitor 320's measuring frequency has been fixed to be slightly above

the serious polarisation region.

35

0

3 8Log Frequency (Hz)

Capacitance (pF)

Electrode polarisation and alpha-dispersion

beta-dispersion

measuring

frequency

fc

Note; In practice these regions overlap

Figure 2 -- Capacitance Vs Frequency

B.5.3 Gas Bubbles

The presence of gas bubbles affects the measured capacitance because they displace some of the yeast

suspension from the sensing region of the probe electrodes. The bubbles contribute very little

capacitance themselves (much less than water) so they always result in a fall in the capacitance reading.


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Research & Development 30/30 Monitoring fermentation with Aber yeast monitor

The fall is proportional to the gas fraction, or "gas break out". Provided this is not abnormally high, and

is reasonably constant, the errors caused are small.

Careful citing of the probe in the yeast or wort main or other pipe work will minimise these effects to

the point where they become insignificant. The Yeast Monitor 320 is also provided with user adjustablesoftware filtering. This can be used to remove any gas break out noise from the instrument reading.

B.5.4 Temperature

There are several different temperature effects.

The background capacitance due to water has a temperature coefficient of -0.5% / degree C which is

equivalent to approximately -0.03pF / degree C. This is quite small compared to typical capacitance

levels encountered in brewery applications (1% spun solids typically gives around 2 pF). As the process

is usually operated at fairly constant temperature, problems rarely occur.

The other effects are due to the ambient temperature experienced by the Yeast Monitor 320 itself, and

that experienced by the Head Amplifier. Again the errors expected are small; the system has a typical

temperature drift of about 0.5 pF for a 30C change in ambient temperature. If the system must be

operated where it will be subject to large temperature swings, particularly when measuring low yeast

concentrations, it is recommended that both the main instrument and the Head Amplifier be protected

by means of suitable enclosures.

Documents

Monitoring Fermentation With an Aber Yeast Monitor