Disc Stack Centrifuge Operating Parameters
and Their Impact on Yeast Physiology
Paul H. Chlup1, Dominic Bernard1 and Graham G. Stewart1,2
ABSTRACT
J. Inst. Brew. 114(1), 45–61, 2008
Hydrodynamic stresses imposed on brewing cells of Saccharo-
myces cerevisiae during beer processing can have a detrimental
impact on beer quality. The use of centrifuges has become an
efficient way to increase brewery throughput as they decrease
clarification times and improve fermenter and tank conditioning
efficiency. The effect of a disc stack centrifuge on yeast and beer
physical stability has been investigated. In this study, a com-
mercial ale yeast strain has been subjected to different operating
conditions during centrifugation. Cell viability and intracellular
pH decreased due to processing conditions encountered during
yeast cropping with a centrifuge. A relationship has been estab-
lished that yeast cell wall mannan, an unfilterable haze con-
stituent, as a function of G-force and centrifugation cycles, is re-
leased from the cell wall while concurrently, particle sizes be-
tween 0.5-2.8 μm and beer haze increased. Furthermore, yeast
intracellular glycogen and trehalose levels were depleted as a
result of centrifugation.
Key words: beer haze, centrifuge, flow cytometry, g-force,
glycogen, intracellular pH, stress, trehalose, yeast.
INTRODUCTION
Economic sustainability procedures have been devel-
oped and implemented by brewing companies due to in-
creasing environmental regulations, consumer pressure
and to demonstrate corporate responsibility. The incentive
to optimize operating costs, while reducing processing
times, in order to gain a competitive advantage and max-
imize revenue, is imperative to brewery survival. Brewers
continuously search for ways to exploit production effi-
ciency36. As a consequence, the disc stack centrifuge has
become a popular component of yeast management sys-
tems in order to reduce fermentation, maturation and clar-
ification times and to control effluent treatment costs.
Modern centrifuges produce forces in excess of 10,000
times the earth’s gravitational constant, achieving solid
separation in seconds with reduced equipment volume.
Centrifuges have a number of applications in a brewery9
and can be used for:
• cropping of non-flocculent yeast cultures at the end
of primary fermentation
1 InternationalCentre for Brewing and Distilling (ICBD), Heriot-
Watt University, Riccarton, Edinburgh, EH14 4AS, Scotland.
2 Corresponding author. E-mail: G.G.Stewart@hw.ac.uk
Publication no. G-2008-0304-554
© 2008 The Institute of Brewing & Distilling
• reducing the yeast quantity from green beer before
the start of secondary fermentation
• beer recovery from cropped yeast
• separation of the hot break after wort boiling
• removal of cold break and yeast at the end of
maturation.
Yeast management and handling systems are influen-
tial in determining the physiological status of yeast, sub-
sequent fermentation performance activity2 and clarifi-
cation during lagering/maturation32. It has been docu-
mented that beer haze can result from the yeast cell wall
releasing mannan as it is processed during agitation in
storage and centrifugation8,19,32,36. These reports suggest
that hydrodynamic stresses have the potential to inflict
damage to the yeast cell wall during beer production and
consequently increase beer haze. However, intermediate
haze formation due to agitation, centrifugation and their
relevant operating parameters still remain of considerable
interest to brewers. During the brewing process, yeast is
subjected to numerous factors that individually or collec-
tively impose stress upon yeast cells. Improperly de-
signed, transfer and environmental factors can cause ther-
mal, osmotic, ethanol, mechanical and shear stresses on
yeast cells1,20,29,33,34,37. A recent investigation conducted by
Kanzleiter16 found that the implementation of newer mod-
el centrifuges along with piping reconfiguration improved
green beer quality compared to an older existing centri-
fuge system.
Although the uses of a centrifuge during brewing are
diverse, this study has concentrated on the effect it has on
yeast and particularly on yeast cell wall damage and the
resulting impact on beer quality and stability. The aim of
this project was to better understand and quantify the ef-
fect of passing Saccharomyces cerevisiae brewing cells
through a disc stack centrifuge. In order to confirm that
the deterioration of yeast was from the centrifugation
process, an excessive number of centrifugation cycles op-
erating at two differing G-forces (high and low) was em-
ployed.
The passage of yeast through the centrifuge exposes
cells to mechanical and hydrodynamic shear stresses8,9,32.
Previous reports have shown2,8,9,17,18,38 that these stresses
can cause a decrease in cell viability and flocculation, cell
wall damage, increased extracellular proteinase A (PrA)
levels, hazier beers and reduced beer foam stability. In
this study, biological indicators of yeast physiology such
as viable and damaged cells, intracellular pH, glycogen
and trehalose levels as well as beer physical stability
parameters including mannan residues, particle size, and
beer haze have been employed to quantify the damage
VOL. 114, NO. 1, 2008 45
which occurs to yeast as a function of cycles through a
centrifuge operating at high and low G-forces. Due to
centrifugal forces within the centrifuge numerous reac-
tionary forces take place between liquid, solid, and discs
including shear stress. Along the bowl wall and support-
ing structures compression and tensile stress are pro-
duced. The forces acting on the disc stack and particle-to-
particle contact are proportional to the G-force. Thus,
operating conditions such as revolutions per minute (rpm)
and flow rate influence forces within the centrifuge.
The flow cytometer is a powerful instrument capable
of determining the physiological status of S. cerevisiae
and aspects of beer stability in near real time11. Flow cy-
tometry possesses technology that performs simultaneous
multiparametic analyses of yeast physical and chemical
characteristics based on cell size, relative granularity and
fluorescence. The particles are transported in a fluid
stream to the interrogation point. The extent to which the
particle scatters light is dependent on the surface topog-
raphy and internal complexity. These characteristics are
determined using a complex optical-to-electronic coupling
system that records the particle’s ability to scatter incident
laser light and the emission of fluorescence15,21,23. The
cells that scatter light are defined by a gate, a numerical or
graphical boundary, the peak mean (Pm) is the relative
fluorescence intensity expressed as the x-mean channel.
The differentiation of subpopulations, based on fluores-
cence intensity, permits the classification of stressed and
non-stressed11 populations of brewing yeasts.
Flow cytometry methods were developed to measure
cell viability, damaged cells, intracellular pH (pHi), man-
nan residues and Pm of intracellular glycogen and tre-
halose11,12. Flow cytometry and fluorescent dyes provide a
rapid and accurate means to quantify the viability and vi-
tality of yeast during centrifugation cycles at differing G-
forces and throughout fermentation. The viability method
developed distinguishes between the dead, alive and dam-
aged cells. The probes propidium iodide (PI) and fluores-
cein diacetate (FDA) have been used as markers to deter-
mine functioning cells. The vitality of the cells was deter-
mined by intracellular pH which employed the pH-
dependent fluorescent probe carboxy SNARF-4F
(SNARF-4F). The ester form of this probe permeates the
cell membrane and once inside the cell, it is cleaved by
esterases to release a pH-sensitive polyanionic probe
which fluoresces44. SNARF-4F possesses two inversely
related emission signals at two different wavelengths,
which makes it possible to calculate the pH from the ratio
between the Pm measured at the two wavelengths44. Yeast
macromolecular components such as intracellular glyco-
gen and trehalose, stained with Schiff reagent and
concanavalin A (ConA) have been assayed by flow
cytometry. In addition, a flow cytometric method has
been developed to detect mannan, an unfilterable haze
constituent, released from the yeast cell wall due to
hydrodynamic stresses during processing3. Flow
cytometry provides an innovative, rapid and viable
option for brewers to evaluate yeast cells throughout the
fermentation cycle and the influence that beer pro-
cessing equipment and operating conditions, in
particular the disc stack centrifuge, have on beer sta-
bility and yeast quality.
46 JOURNAL OF THE INSTITUTE OF BREWING
EXPERIMENTAL
Wort production in the ICBD pilot plant
All malt wort was produced in the ICBD 2 hL pilot
brewery. The malt (Optic) was provided by Pure Malt
(Haddington, UK) and was stored at 11°C before use. The
all malt wort was brewed to a specific gravity of 10°Plato.
The malt was milled with a four hammer ‘Essex Major’
mill (Christy-Hunt, UK) and mashed at a liquor/grist ratio
of 2.5:1. The mashing temperature was maintained at
65°C for 1 h and the temperature was raised to 74°C for
mashing off.
A Meura 2001 42 kg capacity pilot mash filter (Meura,
Belgium) was used for mash separation. The wort was
collected in the kettle and boiled for 1 h achieving 8%
evaporation. Pride of Ringwood hop pellets were used to
provide a beer of 16 IBU’s.
Yeast centrifugation
The yeast culture employed was an ale yeast strain do-
nated by a local commercial brewery. A12°P wort fermen-
tation (2 hL), pitched with 1.2 × 107 cells/mL and a con-
centration of 12 ppm dissolved oxygen, was conducted in
order to have sufficient yeast biomass for passage through
the centrifuge. Two separate regimes through the centri-
fuge were conducted, using an ale yeast strain, at differing
G-forces. Yeast slurry concentrations of 2.5 – 3.0 × 107
cells/mL (2 hL) were cycled through a Westfalia Separator
(Oelde, Germany) SC6-06-076 at the manufacturer’s
suggested flow rate of 5-6 hL/h for this model. The disc
stack height is 150 mm and consists of 76 discs with a
half-cone cone angle of 40°; each disc has an inner radius
of 31 mm and an outer radius of 62 mm. The gap between
the discs is 0.5 mm. The centrifuge bowl speed operated
at 8,000 and 12,000 rpm with an angular velocity of 1047
and 1256 rad/s, which produces 8,900 (low) and 20,000G
(high), respectively, on the outer radius of the disc. The
inlet pressure of the centrifuge was maintained at 0.1 MPa
and the outlet pressure ranged between 0.3-0.4 MPa. Dur-
ing processing, the temperature of the beer was main-
tained via the centrifuge’s heat exchanger. The inlet
temperature of the heat exchanger was 4°C and the outlet
temperature was 5°C.
The yeast was collected by conducting partial dis-
charges of the centrifuge bowl at six minute intervals
during centrifuging and then maintained at 4°C in the col-
lection vessel. During preliminary trials it was determined
that discharging more frequently would dilute the centri-
fuged yeast slurry significantly (process water is used to
flush the bowl). This would make it impractical to re-pitch
the yeast into the 2 L fermentations. A manual discharge
was completed after the centrifugation cycle was com-
pleted. At different operating G-force, yeast samples were
collected, passing through the centrifuge for a total of 9
cycles. Yeast slurry (1 L) was collected at the appropriate
cycle and the cell viability, damaged cells, intracellular
pH, intracellular glycogen and trehalose, mannan, beer
haze and particle size were determined. For fermentation
trials, the yeast exposed to the high G-force was re-cycled
through the centrifuge, to a maximum of 15 cycles, and
subsequently pitched into the appropriate tank. Samples
(700 mL) from the ale yeast were collected at 0 cycle
(control), 7 and 15 cycles for fermentation trials. These
yeast slurries were stored overnight at 4°C prior to re-
pitching into 10°P wort.
Wort fermentation
Fermentations were carried out in triplicate in 2 L
graduated cylinders. The pitching rate was adjusted to
give a viable cell count of approximately 1.0 × 107
cells/mL. Dissolved oxygen concentrations of 10-12 ppm
were introduced into the wort in each 2 L graduated cylin-
der. The fermentation temperature was maintained con-
stant at 21°C. For glycogen and trehalose determinations,
2 mL of fermenting wort was taken by pipetting from the
mid point of the graduated cylinder (800 mL mark). Intra-
cellular glycogen and trehalose were measured throughout
the course of fermentation.
Viability measurements using the
Partec CyFlow SL flow cytometer
Yeast cell viability was measured by the method de-
scribed by Chlup et al.2 Flow cytometric analysis was
performed on the CyFlow SL flow cytometer (manufac-
tured by Partec GmbH, Münster). Data analysis was
performed afterwards using the FloMax software version
2.4e provided by Partec.
Intracellular pH measurements using the
Partec CyFlow SL flow cytometer
A slightly modified version of the method for assessing
intracellular pH in S. cerevisiae cells developed by Valli et
al.44 was used. This method measures the spectral proper-
ties of the probe SNARF-4F (Invitrogen, UK). A 5 mM
stock SNARF-4F solution was prepared from anhydrous
DMSO. The loading buffer (20µM) was made by quanti-
tatively transferring the stock solution with pH 3.0
McIlvaine buffer. Since SNARF-4F is very susceptible to
hydrolysis, the stock solution was used immediately for
sample preparation. For every measurement, the appropri-
ately diluted yeast slurry (< 1000 events/s) sample of 1
mL was collected and centrifuged at 8,000G for 1 min and
re-suspended in 250 µL of loading buffer. After incubation
at 28°C for 11 min on a shaker, the cells were collected by
centrifuging at 8,000G for 1 min and re-suspended in
1000 µL of pH 3.0 McIlvaine buffer. The samples were
placed on ice and analysed by flow cytometry. Each
aforementioned sample was measured 3 times. In situ cal-
ibration must be completed for each experiment as pHi
varies depending on the growth phase of the cells44. Cell
samples were collected and loaded as previously de-
scribed. After reaction with the loading buffer, each sam-
ple pellet was re-suspended in 500 µL of McIlvaine buffer
of varying pH. After addition of 1.5 µL of 9.7 mM am-
photericin B, to final concentration of 30 µM SNARF, the
cells were incubated at 37°C for 1 h on a shaker and then
analysed by flow cytometry. The sample volumes were
adjusted to a final volume of 1000 μL prior to analysis
using the appropriate buffer. All solutions are light sensi-
tive therefore care must be taken to minimise light expo-
sure. All samples and solutions were kept in the dark and
on ice while awaiting analysis. The forward scatter and
side scatter dot plot was used to identify and gate the
yeast population, acquired in log 3 mode, enabled on for-
ward scatter and thresholds set so that cell debris was ex-
cluded from data acquisition. The fluorescence measured
on FL2 (590 nm) and FL3 (680 nm) parameters were ac-
quired in log 4.
Alcohol fixation
The alcoholic fixation enables the permeation of large
dye molecules through the cell membrane. The mem-
branes of living cells are able to selectively mediate the
passage of molecules. Dead cells lack this regulation
function and easily allow substances like large fluores-
cence molecules to enter the cell. The surface structure of
the cells is deformed by means of water removal. The de-
naturation does not alter the composition of protein com-
pounds and many intracellular macromolecules such as
DNA and glycogen are preserved during the alcoholic fix-
ation. Samples of cell suspensions were collected from
the fermentation vessels and were immediately immersed
in 10 mL cold 70% (v/v) ethanol and incubated for at least
24 h at 4°C. Cells treated in this manner can be stored at
4°C for up to one month prior to analysis.
Intracelluar glycogen measurements using
the Partec CyFlow SL flow cytometer
Glycogen staining was carried out using acriflavine ac-
cording to the procedures of Gharton et al.6 and Hutter11
with minor modifications. The Schiff reagent was pre-
pared as follows, diluted HCl solution (1:10) was pre-
pared with distilled water and 15 mL of this dilute acid
was used to dissolve 0.5 g of acriflavine (Sigma, UK).
K2S2O5 (0.5 g) was dissolved in 85 mL distilled H2O. This
K2S2O5 solution was then added to the acriflavine solu-
tion. After 24 h, 0.3 g charcoal was added and filtered.
Sample aliquots (2 mL) of alcohol fixed (70% v/v
EtOH) yeast suspensions were added to 4 mL PBS buffer.
The yeast suspensions were centrifuged for 10 min at
4,000G (10°C). The supernatants were discarded and the
washing step repeated. The resulting pellets were re-sus-
pended in 1 mL periodic acid solution (5 mg/mL of H2O),
vortexed for 10 sec and incubated for 10 min at 20°C.
Samples were then centrifuged at 4,000G for 10 min. The
pellets were re-suspended in 4 mL PBS buffer, centri-
fuged at 4,000G for 10 min and re-suspended in a reaction
chamber with 55 μg/mL Schiff reagent. The suspensions
were incubated for 1 h at 20°C in the dark. After the incu-
bation period, the samples were centrifuged at 4,000G for
10 min (10°C) and the resulting pellets were re-suspended
in 4 mL PBS buffer. This washing step was repeated and
the pellets re-suspended in a final volume of 2 mL in PBS.
The probe was excited using the solid state laser at 488
nm. All data were acquired in log 4 mode and threshold
settings were enabled on forward scatter so that cell
debris was excluded from data acquisition. A total of
30,000 (centrifugation cycles) and 45,000 (fermentations)
events were recorded for analysis. The forward scatter
(FSC) verse side scatter (SSC) dot plot was used to identi-
fy and gate the yeast population. The fluorescence was
measured on FL1 (520 nm) parameter. Each aforemen-
tioned sample was measured 3 times. Data analysis was
performed afterwards using the FloMax software.
VOL. 114, NO. 1, 2008 47
Intracellular trehalose measurements
by the Partec CyFlow SL flow cytometer
The intracellular trehalose content was stained accord-
ing to the method of Hutter et al.12 that utilises the lectin-
fluorochrome-conjugate ConA (Invitrogen, USA). Sample
aliquots (2 mL) of alcohol fixed (70% v/v EtOH) yeast
suspensions were added to 4 mL PBS. The yeast suspen-
sions were centrifuged for 10 min at 4,000G (20°C). The
supernatants were discarded and the washing step repeat-
ed. The resulting pellets were re-suspended in 10 μg/mL
ConA (1 mg/mL of PBS). The suspensions were incubat-
ed for 20 min at 20°C prior to flow cytometric analysis.
All analyses were carried out as previously described for
glycogen staining. A total of 30,000 (centrifugation cy-
cles) and 100,000 (fermentations) events were acquired
for each determination. Each sample was measured 3
times.
Mannan residues determined by the Partec
CyFlow SL flow cytometer
Mannan residues were measured by the method de-
scribed by Chlup et al.3
Mannan and glucose measurements by HPLC
Mannan and glucose was measured by the method de-
scribed by Chlup et al.3
Beer haze
Samples were centrifuged at 730G and degassed. A
Hach 2100N Turbidimeter (Hach Co., Loveland, CO) was
employed for analysis. Each aforesaid sample was meas-
ured 3 times.
Particle size
The particle size and its distribution for all samples
were measured with the Malvern Mastersizer 2000
(Worcestershire, UK) utilizing laser diffraction. Samples
were prepared as described in the mannan residue meth-
od3. Approximately 10 mL of sample was used for each
measurement. The feed rate was set at 2500 and particle
size distribution was recorded. Each abovementioned
sample was measured 3 times.
Fig. 1. Viability and damaged cells as a function of G-force and
centrifugation cycle.
48 JOURNAL OF THE INSTITUTE OF BREWING
Visualization of non-centrifuged and
centrifuged yeast by Environment Scanning
Electron Microscope (ESEM)
Fifty mL aliquots of the appropriate sample were cen-
trifuged at 4°C (730G) for 10 min. The resulting pellet
was frozen at -40°C and then dried for 48 h. The freeze
dried yeast was coated with gold-palladium and subse-
quently reconstituted with water before analysis. Obser-
vations were conducted by a Phillips FEI XL30 FEG-
ESEM. ESEM provides high magnification observation of
yeast cells without damaging the constituents. The ESEM
column was equipped with a multistage differential pres-
sure pumping unit, a pressure of 1-20 torr was maintained
in the observation chamber.
RESULTS AND DISCUSSION
Cell viability and damage
during centrifugation
The data obtained from this investigation validated pre-
vious reports2,8,9 that yeast cells that have undergone cen-
trifugation exhibit lower cell viabilities when compared
with those which had not been centrifuged. This experi-
ment further examined the effects of G-force upon cells as
a function of centrifugation cycle. In order to establish a
relationship between centrifugal G-force and effects on
the physiological status of yeast, it is necessary for yeast
cells to possess an analogous yeast history and physio-
logical state. The yeast strain used in the current study
experienced similar fermentation conditions. The yeast
cell culture exposed to a high G-force underwent a 72 h
conditioning at 10°C and the low G-force yeast was
conditioned for an additional 48 h. Therefore, all cells in
this investigation were under nutrient-limiting conditions
and were in stationary phase.
The high and low G-force yeast cultures were cycled
through the disc stack centrifuge for a total of 9 cycles for
each G-force. The yeast cell viability was measured be-
fore centrifugation and after passage through the centri-
fuge for 3 cycles, 6 cycles, and 9 cycles. The study found,
as would be expected, that yeast cultures exhibited greater
decreases in viability at higher G-force compared to cells
at lower G-force. It has been reported20 that yeast cells in
stationary phase develop thicker cell walls than when in
exponential phase and this increase in cell wall thickness
promotes cell membrane protection and could explain the
increased resilience of the yeast subjected to the lower G-
force. The additional 48 h conditioning period of the low
G-force yeast may have partially contributed to it being
more resilient than the high G-force yeast, but most likely
the hydrodynamic forces within the centrifuge at high G-
force resulted in cell damage and thus a decrease in cell
viability (Fig. 1). Cell wall composition and topography
will vary among different yeast strains37. Therefore, it is
possible that this particular ale strain was naturally more
susceptible to hydrodynamic stresses than other yeast
strains.
It was not possible to confirm from these experiments,
where yeast cell damage occurred within the centrifuge.
Three potential areas where yeast cells experience hydro-
dynamic stresses that decrease viability and vitality are:
(1) centrifuge inlet, (2) in between disc stack gaps, and (3) Beer haze, mannan and particle size
yeast discharge. Cell death results from passive conse- as a function of G-force
quences and degenerative or active processes46. The for-
mer type of cell death is termed necrosis and the latter The impact on the physical damage to yeast cells and
apoptosis46. Necrosis is a consequence of passive cell consequent physical beer stability is dependent on centri-
injury. Alternatively, apoptosis is driven by a complex fuge operating parameters. Hydrodynamic forces and
process known as programmed cell death. Morphological yeast cell interaction within the gap of the disc stack cre-
and physiological consequences resulting from necrosis ates collisions among the yeast cells producing kinetic en-
differ from apoptosis. The differences mainly concern cell ergy causing cellular damage. Increasing G-force will ex-
shape and cell structural features. pose yeast cells to detrimental effects of hydrodynamic
Flow cytometry viability analysis discriminates be- forces. Release of mannan during mechanical agitation of
tween necrotic and apoptotic cells allowing for quantifica- yeast slurries in conjunction with an increase in beer haze
tion and identification of populations. The viability assay has been previously documented3,19,40. It has been reported
differentiated between dead, living, and damaged cell that beer haze can result from the yeast cell wall releasing
populations stained with FDA and PI, quantifying yeast mannan2,8,35,36 as it is processed during centrifugation. Ad-
sub populations. Cell esterases, capable of hydrolyzing ditionally, several reports have confirmed mannan, which
FDA to fluorescein, indicated living cells, and PI, a nu- is unfilterable from the medium, to be a constituent of
cleic acid stain that permeates compromised membranes, haze material19,35,38. Haze was derived from pieces of
distinguished dead cells from live ones. Damaged cells material released from the yeast wall exterior as cells
stain with both FDA and PI. It was determined that passed through the centrifuge. Moreover, McCourtie and
necrotic and apoptotic cells represent damaged and dead Douglas22 determined that Candida albicans cell wall
cells, respectively. composition and adherence are a function of environmen-
At the end of 9 cycles, with the culture exposed to high tal conditions, which implies that S. cerevisiae is similarly
G-force during centrifugation, viability decreased by prone to damage inflicted by stressful treatment4. ESEM
92.4% and following exposure to low G-force by 79.6% analysis (Figs. 2A and B) provided visual evidence of
(Fig. 1). Additionally, a larger percentage of yeast cells yeast cell damage and the release of cellular wall compo-
subjected to low G-force (37.5% compared with 2.5% for nents as a result of disc stack centrifugation at high G-
high G-force) were associated with damaged cells. The force.
higher proportion of damaged cells of low G-force yeast
culture compared to high G-force implies that these cells
are still able to function and are more resilient to centrifu-
gal forces whereas the low proportion of damaged cells at
high G-force remains constant indicating a permanent loss
of functioning cells. These results suggest that yeast cells
as a function of G-force and centrifuge cycle, experience
a damage cell threshold (Fig. 1). Damage cell threshold,
the transition from the necrotic to the apoptotic cell state,
is encountered when the centrifugation hydrodynamic
stress is sufficient to cause death to the cell. A damaged
cell’s capacity to function or proliferate, after exposure to
hydrodynamic stresses, is not fully understood and re-
quires further investigation.
Both yeast cell cultures demonstrated the same general
trend of a decrease in viability as a function of centrifuga-
tion cycle. At high G-force, the viability decreased by
83% (3 cycle), 47% (6 cycle), 12% (9 cycle) whereas the
low G-force decreased by 41% (3 cycle), 25% (6 cycle),
and 52% (9 cycle). The percentage of damaged cells for
low G-force culture increased by 34% between the control
and cycle 3, then decreased 7% between cycle 3 and 6 and
a further reduction of 7% between cycle 6 and 9 was ob-
served. The percentage of damaged cells for the high G-
force strain was fairly consistent (1 – 2.5%), throughout
centrifugation cycles. These results provide evidence that
the amount of G-force applied to yeast cells during centri-
fugation contributes to a decrease in viability and influ-
ences the damaged cell threshold. However, because only
one yeast strain was studied, it is necessary to conduct
further testing that considers additional yeast strains, their
history and physiological status in order to develop a ho- Fig. 2. (A) Yeast cells before passage through a disc centri-
listic theory of the effects of yeast centrifugation on beer fugation or exposure to high G-forces. (B) Yeast cells following
quality. passage through a disc centrifugation at a high G-force.

VOL. 114, NO. 1, 2008 49

 The impact that G-force has on beer haze and mannan
residues has been investigated (Figs. 3 and 4). Beer haze
increased following both high and low G-force, compared
to the control and haze increased 32.9 EBC or 264% (cycle
3), 25.2 EBC or 202% (cycle 6) and 27.9 EBC or 224%
with high G-force. The increase in beer haze at the low G-
force was not as extreme compared to the control, as in-
creases of 5.7 EBC or 16% (cycle 3), 4.6 EBC or 17% and
4.0 EBC or 15% (cycle 9) were observed. Mannan residues
increased as a function of high G-force compared to the
control, where increases of 8.5 × 105 or 106% (cycle 3), 1.5
× 105 or 18% (cycle 6), and 4.1 × 105 or 51% (cycle 9) were
observed. Initial increase in mannan residues was antici-
pated but the subsequent decline during the later cycles was
not expected. Williams and Wiseman43 showed that a man-
nan-enzyme complex from the cells was released by os-
motic shock. Moreover, osmotic shock and shear stress
caused the loss of cell surface fimbriae and flocculation4.
Thus, the high G-force promoted a large portion of the
mannan to be broken off initially and the continuous cy-
cling through the centrifuge facilitated efficient separation
consequently reducing both beer haze and mannan resi-
dues. However, reduction below the original mannan resi-
due concentration was not achieved. In contrast, at low G-
force, mannan residues decreased compared to the control, in
which a reduction of 9.6 × 105 or 35% (cycle 3), 8.5 × 105 or
43% (cycle 6), 8.5 × 105 or 50% (cycle 9) was discernible.
These data suggest, as would be expected, that at low G-
force, yeast cells are resistant to hydrodynamic stress.
Fig. 3. Haze and mannan residues as a function of high G-force
and centrifugation cycle.
Fig. 4. Haze and mannan residues as a function of low G-force
and centrifugation cycle.
50 JOURNAL OF THE INSTITUTE OF BREWING
The particle size analysis gave further insight into the
extent of yeast cell damage produced by centrifugation
(Figs. 5A and B). Particles sizes ranging between 0.02 and
5.02 μm were measured after the sample had been
concentrated by ultraspeed centrifugation. Figs. 5A and B
illustrate the distribution of particle sizes expressed in
percentage. Particle analysis for high G-force clearly indi-
cates size increases between 1.0-2.8 μm as yeast was
passed through the centrifuge. Particle sizes between
0.56-1.4 μm increased, compared to the control, increases
of 0.8 or 60% for cycle 3, 0.5 or 39% for cycle 6 and 2.7
or 202% for cycle 9 were observed. Additionally, there
was a rise in particle size between 1.4-2.8 μm, increases
of 2.2 or 49% (cycle 3), 1.5 or 32% (cycle 6) and 4.9 or
110% (cycle 9) was observed. Particle sizes between 2.8-
5.0 μm exhibited a dissimilar pattern, reductions of 4.4 or
37% particle volume (cycle 3), 3.2 or 27% (cycle 6) and
1.6 or 14% (cycle 9) were detected.
At low G-force, there was a minimal particle size fluc-
tuation between 0.56-2.8 μm, except particle sizes be-
tween 1.4-2.8 μm for cycle 9 increased 3.8 or 38%. Parti-
cle sizes between 2.8-5.0 μm decreased as a function of
centrifugation cycle number compared to the control, de-
creases of 0.6 or 3% (cycle 3), 3.5 or 20% (cycle 6) and
8.0 or 44% (cycle 9) have been observed. Yeast wall
breakage at high G-force was proportionally larger as
demonstrated by increasing particle size between 0.56-2.8
μm which ultimately contributes to beer haze. Contrary to
high G-force, the data shows evidence that yeast cells sub-
jected to low G-force did not result in yeast cell breakage
as particle size did not increase. Particle size for low G-
force (Fig. 5B) were > 2%, whereas high G-force (Fig.
5A) particle size were < 2%. This implies that separation
is more efficient at high G-force because smaller particle
size percentages were observed.
Quantification of mannose/glucose ratio
by HPLC
Malcorps et al.21 reported that intracellular glycogen
particles are released during primary fermentation due to
stressful conditions, which can cause an unfilterable haze.
Thus, the sugar composition of the supernatant before and
after centrifugation was analysed, following acid hydroly-
sis, by HPLC to determine the mannose and glucose con-
centrations2. The HPLC results obtained from this study
determined that glucose levels in the supernatant did not
increase after the yeast slurry had been centrifuged at ei-
ther high or low G-force (Fig. 6). This indicates that the
hydrodynamic forces caused by centrifugation in these ex-
periments did not cause release of glycogen particles. Ad-
ditionally, the mannose/glucose ratio increased as the
number of centrifugation cycles increased (Fig. 7), con-
firming the flow cytometer results that hydrodynamic
forces elicited the release of mannan residues from the
yeast cell wall and not glycogen particles from the yeast
cytoplasm.
Intracelluar pH measurement
by flow cytometry
The intracellular pH (pHi) measurements were based
on the assay described by Valli et al.44 The method obtains
the pHi distribution within a cell population based on the
 Fig. 5. (A) Particle size (%) as a function of high G-force and centrifugation
cycle. (B) Particle size (%) as a function of low G-force and centrifugation cycle.

Fig. 6. Glucose concentrations of yeast slurry supernatants cen-

trifuged at high and low G-forces determined by HPLC analysis
pH dependent emission spectra of the probe SNARF-4F.
A ratiometric calculation based on the inversely related
emission signals, the Pm expressed as x-mean channel, at
590 nm and 680 nm determines pHi. The pHi dependent
shift is illustrated with unstained and cells stained with
SNARF-4F (Figs. 8A and B). The yeast cell population
was gated in the FSC versus SSC dot plot. A threshold
was defined to eliminate debris in the sample.
Fig. 7. Mannose/glucose ratio of centrifuge yeast slurry super-
natant determined by HPLC analysis.
Intracelluar glycogen measurement
by flow cytometry
Various trials were conducted using unstained cells and
cells stained with Schiff reagent. The data generated were
used to determine where the viable/depleted cell popula-

VOL. 114, NO. 1, 2008 51

 Fig. 8. (A) The peaks represent the autofluorescence and debris of an unstained Intracellular pH
sample. Alternatively, stained cells with SNARF exhibits a significant pH-dependent emission shift
from yellow-orange to red fluorescence under acidic and basic conditions, thus, allowing the ratio
of the fluorescence intensities at two emission wavelengths to be used for quantitative determina-
tions of intracellular pH. (B) SNARF-4F stained cells with pH dependent shift observed. SNARF-
4F dye is loaded into the sample as cell-permeant AM ester acetate. The ester form of this probe
permeates the cell membrane, once inside the cell it is cleaved by esterases to release a pH sensitive
polyanionic probe which fluoresces. SNARF-4F undergoes a shift to longer wavelengths upon de-
protonation of its phenolic substituent. The pH-dependent spectral shifts exhibited by SNARF-4F
allows for in vitro calibration of the pH response in terms of the ratio of fluorescence intensities
measured at two different wavelengths, typically 590 nm and 680 nm, and excitation at 488 nm.

tions and debris appeared on the histograms and dot plots
generated by the flow cytometer. Figs. 9A, B and C dem-
onstrate the fluorescence produced by the Schiff reagent
and how these patterns can be used to separate viable and
depleted cell populations. Gating was used to define the
yeast cell population. Data from within this population
can be plotted on dot plots and histograms for further
analysis. A single parameter histogram views one param-
eter against the number of events, whereas dot plots with
range markers display the fluorescence data from events
in the gated region. The yeast cell population was gated in
the FSC versus SSC dot plot. The gating on the FSC and
SSC determined the viable and depleted cells and the Pm
characterised. Yeast cells stained with Schiff reagent fluo-
resced at 520 nm (FL1). The threshold was adjusted such
that the debris and autofluorescence of the unstained cells
were not included in the measurement.
Intracelluar trehalose measurement
by flow cytometry
The method for the assessment of intracellular treha-
lose content was based on the method described by Hutter

52 JOURNAL OF THE INSTITUTE OF BREWING

et al.12 Analysis was conducted using unstained cells and
cells stained with ConA. These examinations were used to
establish where the viable/depleted cell populations and
debris are discernible on the histograms and dot plots gen-
erated by the flow cytometer. The fluorescence produced
by ConA is illustrated and how these patterns can distin-
guish viable and depleted cell populations (Figs. 10A, B
and C). Gating and instrument settings were similar to
those described for intracellular glycogen determination.
Intracelluar pH during centrifugation
Research on yeast physiology has indicated that the
cell’s ability to maintain and regulate its pHi is crucial to
ensure proper functioning of cellular processes44. The pHi
in yeast cells is regulated by the plasma membrane
ATPase, which acts as a proton (H+) pump14. This proton
pump plays a significant role in cell proliferation activity
and fermentation regulation10. The transmembrane H+ gra-
dient, which is generated, is the driving force for the
transport of nutrients (for example, maltose and amino ac-
ids) across the cell membrane44. The pHi homeostasis is
very important, as key enzymes in glycolysis and gluco-
neogenesis are regulated by cascade reactions which are
pH dependent (c-AMP dependent protein kinases; phos-
phorylase, glycogen synthase, trehalase, fructose-1,6-bis-
phosphatase, 6-phosphofructo-2-kinase)14. Imai et al.14
found that under acidic conditions, cells possessing a high
H+ extrusion activity were more vital than cells having a
low extrusion activity.
The findings from these experiments established that

Fig. 9. (A) Intracellular glycogen of control yeast cells (prior to centrifugation) stained by Schiff reagent. A bimodal cell population
was observed. The viable cells (R1) fluoresce more intensely than depleted cells (R2). The viable cells are larger in size and have a
different internal granularity compared to depleted cells. A bimodal population implies yeast with differing physiological conditions
were present in the cell population. (B) Glycogen peak mean (Pm) of viable cells. These cells possess higher proliferation capacity than
depleted cells. A fraction of this population is mother and daughter cells, which potentially have a larger glycogen content. A Pm
increase was observed during anaerobic and nutrient rich conditions. (C) Depleted cells glycogen peak mean (Pm) represents the
population with lower glycogen levels, proliferation capabilities and exhausted cells. Lower glycogen levels imply decreased
fermentation activity of the cell population. (D) A confirmation of the bimodal distribution of glycogen content in the control cell
population. 3D plot generated by WinMDI software (http://facs.scripps.edu/software.html).

VOL. 114, NO. 1, 2008 53

the pHi of yeast cells and viability was interrelated during
centrifugation cycles. Additionally, the pHi was dependent
on the amount of G-force applied during centrifugation
(Fig. 11A). Initially, the pHi of the high G-force yeast
slurry had a higher pHi compared to the low G-force yeast
slurry, a difference of 0.25 or 4.5% is observed, which
may have resulted from the extra 48 h conditioning period.
The data revealed that upon centrifugation, the high G-
force yeast slurry pHi decreased, whereas, the low G-force
yeast slurry increased. This trend continued throughout
the duration of centrifugation. During the first 3 centrifu-
gation cycles the pHi of the low G-force yeast slurry was
0.54 pHi or 9.9% higher than the high G-force. For cycle
6, the pHi difference between the G-forces was less
dramatic; the low G-force slurry experienced a 0.36 pHi or
6.5% increase compared to the high G-force. A difference
of 0.67 pHi or 12.5% was observed at cycle 9 between
high and low G-force. In agreement with Imai and Ohno13,
these results provide a correlation between decreasing pHi
and increasing numbers of dead cells. Consequently, the
pHi provides insight into yeast physiology and indicates
yeast cell functionality loss due to high G-force operating
conditions encountered when passed through a disc stack
centrifuge.

Fig. 10. (A) Intracellular trehalose of control yeast cells stained by ConA. Similar to intracellular glycogen, a bimodal cell population
was observed. The viable cells (R1) fluoresced more intensely than depleted cells (R2). The two distinctive fluorescent properties
represent subpopulations within the pitching yeast. (B) Trehalose peak mean (Pm) of viable cells. Trehalose is a cell wall constituent
comprised of α-glucopyranose and is an osmoprotectant. The viable cell population Pm has a low trehalose level at the beginning of
fermentation. Trehalose Pm level increases at the beginning phase of proliferation signifying the yeast’s adaptation to a new environ-
ment. (C) Trehalose peak mean (Pm) of depleted cells. Environmental factors such as the depletion of nutrients, high ethanol and CO2
concentrations coincide with an increase in trehalose content. High trehalose concentrations indicate the cells’ ability to withstand
stressful conditions during fermentation. (D) Bimodal distribution of trehalose in the control cell population. 3D plot generated by
WinMDI software. There is a higher proportion of depleted cells with higher fluorescence intensity content at 0 h compared to the via-
ble cells. This may indicate that these cells are quiescent.

54 JOURNAL OF THE INSTITUTE OF BREWING

Intracellular glycogen at two
different operating G-forces
The use of yeast cell glycogen content as a sensitive
parameter to evaluate physiological status and fermenta-
tion performance has been well documented by many
brewing scientists1,11,24,25,30,37. Glycogen serves as a reserve
carbohydrate and promotes the synthesis of lipids during
aerobic conditions, stimulating cell membrane structural
integrity25. Quain26 established three distinct phases of
metabolic activity of yeast intracellular glycogen through-
out fermentation, consisting of an initial rapid decrease in
aerobic conditions, accumulation during the lag and early
exponential phase of fermentations when wort nutrients

Fig. 11. (A) Intracellular pH as a function of G-force and centrifugation cycle. (B)
Intracellular glycogen as a function of G-force and centrifugation cycle. (C) Intra-
cellular trehalose as a function of G-force and centrifugation cycle.

VOL. 114, NO. 1, 2008 55

are available, and glycogen reduction during the station-
ary phase. Stewart and Russell37 confirmed Quain and
Tubb’s27 report while demonstrating a relationship between
intracellular glycogen reduction and nutrient exhaustion
during fermentation. An elevated level of glycogen util-
ization during stationary phase reduces the opportunity of
cell proliferation in subsequent fermentations37. Further-
more, the intracellular glycogen level in the stationary
phase cells is a function of yeast strain, fermentation
conditions, and wort gravity37.
The use of flow cytometry has shown11 that fermenting
yeast populations consist of different glycogen levels and
physiological states. Intracellular glycogen was measured
over the course of the centrifugation cycles at two differ-
ent G-forces (Fig. 11B). The results show that centrifuged
yeast exhibited higher levels of intracellular glycogen
compared to non-centrifuged yeast. It is plausible to at-
tribute this unexpected result to the use of compressed air
to transfer the yeast slurry from the holding vessel to the
disc stack centrifuge. Additionally, care was not taken
throughout the experiment to purge the centrifugation cir-
cuit with CO2. Agitation of the yeast culture during mix-
ing and transfer coupled with an aerobic atmosphere pro-
vided the opportunity for dissolved oxygen assimilation.
The results obtained from this experiment confirm pre-
vious reports28,37 that yeast cells in stationary phase exhib-
ited low intracellular glycogen concentrations compared
to exponential phase cells (Figs. 11B and 12). The peak
mean (Pm) of the high and low G-force control possessed
similar values of 6.0 and 9.2. It was observed that the low
G-force centrifuged yeast increased to 55.2 Pm or 497%
after cycle 3, for cycle 6 increased 66.0 Pm or 19.2% and
cycle 9 increased to 68.1 Pm or 3.1%. An alternative trend
was observed for the high G-force yeast which had a mar-
ginal increase of 9.4 Pm or 54% after cycle 3, followed by
a cycle 6 increase of 50.1 Pm or 436% and a cycle 9 in-
crease of 1.9 Pm or 3% was observed.
Aforementioned reasons may have contributed to the
increase in intracellular glycogen; unfortunately, dis-
solved oxygen was not measured during the course of the
centrifugation cycles. Consequently, it is difficult to posi-
tively state that the increase in glycogen content was a re-

Fig. 12. (A) Intracellular glycogen peak mean (Pm) of viable cells as a function G-
force and fermentation time. (B) Intracellular glycogen peak mean (Pm) of depleted
cells as a function of G-force and fermentation time.

56 JOURNAL OF THE INSTITUTE OF BREWING

sult of oxygen assimilation. Additionally, investigations
by Slaughter and Nomura33 found that decreased viability
did not necessarily correlate with a decline in glycogen
content, which was confirmed in this study.
Intracellular glycogen levels
during fermentation
Fermentation trials were conducted with yeast that had
been passed through the centrifuge for 15 cycles at high
G-force. A bimodal distribution of glycogen relative fluo-
rescence intensity was observed indicating that within the
yeast population there were two populations with quantifi-
able physiological states (Figs. 9A and D). R2 represents
the depleted cell population (Fig. 9C). Additionally, the
viable cells in the second peak possess higher prolifera-
tion capacity (Fig. 9B).
The bimodal distribution was observed throughout the
fermentations. Although, at the beginning of fermentation,
centrifuged yeast cells possessed larger depleted cell pop-
ulations. The fact that centrifuged yeast did not show the
same bimodal distribution (Figs. 13A, B and C) as non-
centrifuged yeast could be a result of newborn and senes-
cent cells being damaged during the centrifugation pro-
cess. Therefore, these yeast cells were not quantified in
the viable cell population. Furthermore, these cells could
have been predisposed to necrosis in the centrifuge result-
ing in loss of functionality.
Figs. 12A and B provide results that establish that gly-
cogen levels oscillate during the first 48 h of fermentation
which confirms the Hutter11 findings. It has been report-
ed26,28,37 that as glycogen is metabolised during the early
stages of fermentation, for the synthesis of lipids one
would anticipate an initial reduction followed by an in-
crease as the yeast undergoes regeneration of its glycogen
levels. The anticipated decrease in glycogen levels at the
start of fermentation was not observed. The discrepancy
observed between the results obtained by flow cytometry
and those reported by conventional glycogen determina-
tions requires further investigation. In a comparison of the
viable cell population of control to cycle 7, differences of
27 Pm or 16% (0 h), 23 Pm or 8% (8 h), 89 Pm or 34% (28
h) and 86 Pm or 36% (48 h) were observed. Similarly,

Fig. 13. (A) Intracellular glycogen of yeast cells after 7 cycles through the centrifuge. These cells
represent a decrease in functionality of the cell population. Due to stress resulting from high G-
force the depleted cell glycogen content increases. (B) A decrease in glycogen peak mean (Pm) of
viable cells due to passage through a centrifuge results in the cell population’s ability to proliferate
decreasing. (C) Glycogen peak mean (Pm) of depleted cells has increased proportionally to viable
cells after centrifugation. These cells are newborn and senescent cells being destroyed during the
centrifugation process and thus eliminated from the viable cell population.

VOL. 114, NO. 1, 2008 57

comparing viable cell populations of control to cycle 15,
differences of 6 Pm or 3% (0 h), 41 Pm or 13% (8 h), 96 Pm
or 34% (28 h) and 167 Pm or 51% (48 h) were shown.
After 48 h fermentation, the glycogen declined steadily
throughout the course of fermentation. There were only
marginal differences of glycogen Pm between cycle 7 and
cycle 15.
The glycogen Pm displayed a similar trend for the de-
pleted cells, when comparing the control to cycle 7, differ-
ences of 9.5 Pm or 10% (0 h), 66 Pm or 44% (8 h), 60 Pm or
36% (28) and 43 Pm or 27% (48 h) were found. Evaluation
of the control and cycle 15 glycogen Pm, differences of 6.3
Pm or 6% (0 h), 74 Pm or 47% (8 h), 67 Pm or 40% (28 h)
and 89 Pm or 58% (48 h) were revealed. Correspondingly,
only minor fluctuations of glycogen Pm existed between
cycle 7 and cycle 15. In the study by Shen et al.31, a cor-
relation between diminished glycogen content in immobi-
lized cell systems compared to a free cell system speculat-
ed that lower glycogen levels were attributed to counter-
acting stresses. The same investigation concluded that
immobilized yeast cells were not able to accumulate as
much glycogen as free cells. This data provides verifica-
tion that hydrodynamic stresses have potential to unfa-
vourably affect yeast cell physiology. Alternatively, Doran
Fig. 14. (A) Intracellular trehalose peak mean (Pm) of viable cells as a function G-
force and fermentation time. (B) Intracellular trehalose peak mean of depleted cells as
a function of G-force and fermentation time.
58 JOURNAL OF THE INSTITUTE OF BREWING
and Bailey5 reported that immobilized yeast had a higher
macromolecule composition than free cell systems. The
data from the fermentation trials displayed a clear rela-
tionship between depleted glycogen Pm levels as a func-
tion of centrifugation cycle (Figs. 12A and B). Therefore,
centrifuged yeast cells may have insufficient glycogen to
“fuel” lipid metabolism thus restricting cell growth and
fermentation performance.
Intracelluar trehalose at two
different operating G-forces
Stewart and Russell37, Slaughter and Nomura33 and
Majara et al.20 have presented evidence that intracellular
trehalose levels increased as a response to stress. The role
of intracellular trehalose as a yeast cell membrane stabi-
lizer has been reported by Thevelein39. Majara et al.20
found that cellular trehalose levels accumulated propor-
tionally to wort density indicating that it serves as an os-
moprotectant during high gravity wort fermentations. Ad-
ditionally, yeast cells with elevated levels of intracellular
trehalose possess increased osmotolerance, thermotoler-
ance, and ethanol tolerance37,45. Therefore, high levels of
intracellular trehalose are associated with yeast cells that
are resistant to stressful conditions.
 The degree of stress resistance and the amount of tre-
halose synthesized are dependent on the physiological
state of the yeast cultures. Non-growing cells were shown
to be more stress resistant, whereas growing cells pro-
duced more trehalose in response to the applied stress.
Guldfeldt and Arneborg7 indicated that increased levels of
trehalose in pitching yeast sustained high cell viability
during the initial stages of fermentation and resulted in
higher carbohydrate utilisation rates and increased pro-
duction of isoamyl alcohol and iso-butanol. Alternatively,
procedures that affect membrane integrity such as autoly-
sis, heating, drying and treatment with organic solvents,
caused rapid trehalose degradation34,41. Neutral trehalases,
which mediate the degradation of trehalose42, are activated
and result from a rapid increase in cAMP levels in yeast39.
The abovementioned research indicates that trehalose
levels may be useful in evaluating yeast vitality. Boulton1
found that the degradation of trehalose correlates with a
rapid loss of viability1,33. The data obtained from these ex-
periments (Fig11C) confirm these findings. The trehalose
Pm of the high and low G-force control possess Pm values
of 67 and 95, respectively. The higher level of trehalose
Pm might be partially attributed to the additional 48 h
yeast conditioning period. Trehalose Pm decreased as a
function of centrifugation cycle, compared to the control.
High centrifuge G-force yeast, resulted in trehalose reduc-
tions compared to the control of 53.4 or 21% (cycle 3),
22.1 or 67% (cycle 6) and 31.8 or 58% (cycle 9) were
determined. A similar diminishing trehalose Pm trend was
observed for the low G-force yeast, compared to the con-
trol, decreases of 26.5 or 72% (cycle 3), 30.9 or 67%
(cycle 6), 33.1 or 66% (cycle 9) were observed. Low tre-
halose content reflects low stress tolerance of centrifuged
yeast cells. Shen et al.31 found a similar reduction of intra-
cellular trehalose in immobilized yeast systems and
Slaughter and Nomura33 described a similar pattern during
stress induced fermentations.
Intracellular trehalose levels
during fermentation
As previously mentioned, the intracellular concentra-
tion of trehalose has been reported20 to play an important
part in the cells’ ability to withstand adverse environmen-
tal conditions. Fermentation trials were conducted with
yeast that had been passed through the centrifuge for 15
cycles at high G-force (Figs. 14A and B). Throughout the
fermentation cycle, a bimodal distribution of trehalose Pm
was observed indicating that within the yeast population
there were analogous populations with different physio-
logical characteristics (Figs.10A and D). R2 represents the
depleted cell population (Fig. 10C) which has a lower
trehalose Pm. Moreover, the viable cells in the second
peak contained a higher trehalose Pm (Fig. 10B). Alterna-
tively, centrifuged yeast cells displayed higher levels of
depleted trehalose Pm (Figs. 15A, B and C).
The initial elevated trehalose Pm levels would seem to
substantiate the findings of Majara et al.20 and Pratt-
Marshall22 that trehalose concentrations were higher at the
beginning of fermentation. Consequently, compared to the
control, at 0 h, viable yeast cells during cycle 7 and 15 de-
creased by the same amount of 241 Pm or confirmed simi-
lar findings of Rambeck and Simon41, Souza and Panek34
and Shen et al.31 At 8 h when compared to the control, vi-
able cells trehalose Pm level had a difference of 114 Pm or
36% (cycle 7) and 98 Pm or 31% (cycle 15) with the de-
pleted cells 114 Pm or 36% and 111 Pm or 57%, respec-
tively. This indicates a rise in trehalose Pm for the centri-
fuged cells, although not to the degree of the control.
Pratt-Marshall24 reported that ale yeast fermenting in 20o
Plato wort exhibited higher trehalose levels than the same
yeast fermenting in 12o Plato wort and that the yeast fer-
menting in the high gravity wort showed a trehalose peak
after 24 h of fermentation. Although depleted cells did not
depict this peak, the viable cells population of centrifuged
yeast (cycle 7 and 15) had a comparable peak after ap-
proximately 16 h of fermentation. These findings are in
agreement with the results of Pratt-Marshall24, where
stressed cells maintained higher trehalose levels towards
the end of fermentation.
CONCLUSIONS
The objective of these experiments was to determine
whether the passage of a brewers’ yeast strain through a
disc stack centrifuge at differing operating G-force condi-
tions had a negative effect on cell viability, vitality and
beer physical stability. Yeast cropping by centrifugation
exposes yeast to extreme shear stress. The viability of the
yeast culture tested decreased as it was re-cycled through
the centrifuge. There were proportionally higher percent-
ages of damaged yeast cells that were exposed to the low
G-force implying that these cells were more resistant to
hydrodynamic forces during centrifugation. The results in
Fig. 1 demonstrate the damage cell threshold, the transi-
tion from the necrotic to the apoptotic cell state, due to
exposure to hydrodynamic stresses within the centrifuge.
The results also provided evidence that the beer haze in-
creased, mannan residues were produced, and particle size
of < 2.8 µm increased as exposure to high G-force in the
centrifuge was applied. The physiological state of the
yeast was modified due to the processing conditions en-
countered during yeast cropping. Glycogen and trehalose
levels were depleted. These changes in cellular properties
influence metabolic activity and reduce stress resistance.
The flow cytometer allows for a graphic representation of
the different subsets within a yeast population, and it can
be used to perform various analyses such as cell viability,
damaged cells, intracellular pH, mannan residues and the
determination viable and depleted intracellular macromol-
ecules. During these experiments, the centrifuge was op-
erated under conditions similar to those encountered dur-
ing commercial production. However, shear stresses may
occur with or without the use of a centrifuge, leading to
decreased beer quality and stability. It is important for
yeast management systems that utilize centrifuges be de-
signed and operated properly to minimize negative conse-
quences on beer quality. Recent investigations found that
implementation of newer model centrifuges and rede-
signed pipe work improved green beer quality. From this
experiment, it can be concluded that the passage of yeast
cells through a disc stack centrifuge, at high G-force,
strongly adversely influences yeast physiology and beer
physical stability.
VOL. 114, NO. 1, 2008 59
 Fig. 15. (A) Intracellular trehalose of yeast cells following 7 cycles through the centrifuge. De-
pleted cells increased proportionally compared to viable cells. (B) Trehalose peak mean of
viable cells. A large decrease in viable cells is observed after centrifugation. These cells do not
possess stress resistant attributes which may have an affect on fermentation performance and
subsequent repitching. (C) Trehalose peak mean of depleted cells. These cells represent the pop-
ulation which has been passed through the centrifuge. There is a larger degree of depleted cells
with higher trehalose content, which implies these cells were stressed as a function of passage
through the centrifuge.

ACKNOWLEDGMENTS tion of Saccharomyces cerevisiae attached to gelatin. Bio-

We thank the IBD Grants Committee for financial support.
We also thank Graham McKernan for assistance in the pilot
brewery, Jim MacKinlay for assistance with the particle size
analysis and Dr. Jim Buckman for support with the Environmen-
tal Scanning Electron Microscopy.
LITERATURE CITED
1. Boulton, C., Trehalose, glycogen and sterol. In: Brewing Yeast
Fermentation Performance, K.A. Smart, Ed., Blackwell Sci-
ence: London, 2000, pp.10-19.
2. Chlup, P.H., Bernard, D. and Stewart, G.G., The disc stack cen-
trifuge and its impact on yeast and beer quality. J. Am. Soc.
Brew. Chem., 2007, 65, 29-37.
3. Chlup, P.H., Conery, J. and Stewart, G.G., Detection of mannan
from Saccharomyces cerevisiae by flow cytometry. J. Am. Soc.
Brew. Chem., 2007, 65, 151-156.
4. Day, A.W., Poon, N.H. and Stewart, G.G., Fungal fimbriae. III.
The effect on flocculation in Saccharomyces. Can. J. Microbiol.,
1975, 21, 558-564.
5. Doran, P.M. and Bailey, J.E., Effects of immobilization on
growth, fermentation properties, and macromolecular composi-
technol. Bioeng., 1986, 28, 73-87.
6. Gharton, G., Olsson, I. and Dahlqvist, A., Determination of the
glycogen content in single neutrophil leucocytes using micro-
model of leucocytes glycogen. J. Histochem. Cytochem., 1975,
23, 59-64.
7. Guldfeldt, L.U. and Arneborg, N., The effect of yeast trehalose
content at pitching on performance during brewing fermen-
tations. J. Inst. Brew., 1998, 104, 37-39.
8. Harrison, S.T.L., Basson, L., Robinson, A., Godfrey, T.A.,
O’Connor-Cox, E. and Axcell, B., Mechanical handling of
brewers’ yeast during cropping and its effects on yeast quality.
Proceedings of the 6th Institute of Brewing Convention (Central
and Southern Africa Section), 1997, pp.55-60.
9. Harrison, S.T.L. and Robinson, A., Disc stack centrifugation of
the recovery of the brewers’yeast – Its effect on yeast cell sur-
face, flocculation and fermentation performance. Proceedings of
the 8th Institute of Brewing Convention (Africa Section), 2001,
pp.157-164.
10. Heggart, H.M., Margaritis, A., Pilkington, H., Stewart, R.J.,
Sobczak, J. and Russell, I., Measurement of brewing yeast via-
bility and vitality: a review of methods, Tech. Q. Master Brew.
Assoc. Am., 2000, 37, 409-430.
11. Hutter, K.J., Flow cytometry–A new tool for direct control of

60 JOURNAL OF THE INSTITUTE OF BREWING

 fermentation process. J. Inst. Brew., 2002, 108, 52-53.
12. Hutter, K.-J., Kliemt, C., Nitzsche, F. and Wießler, M., Bio-
monitoring der Betriebshefen in Praxi mit Fluoreszenzoptischen
Verfahren. IX. Mitteilung: Trehalose – Streßprotektant der Sac-
charomyces-Hefen. Brauwissenschaft, 2003, 56, 121–125.
13. Imai, T. and Ohno, T., The relationship between viability and in-
tracellular pH in yeast Saccharomyces cerevisiae. Appl. Environ.
Microbiol., 1995, 61, 3604-3608.
14. Imai, T., Nakajima, I. and Ohno, T., Development of a new
method for the evaluation of yeast vitality by measuring intra-
cellular pH. J. Am. Soc. Brew. Chem., 1994, 52, 5-8.
15. Introduction to Flow Cytometry: A Learning Guide Manual Part
Number: 11-11032-01 April 2000. http://www.stemcell.umn.
edu/img/assets/10061/Intro_to_Flow_Cytometry_Learning_Gui
de.pdf (last accessed January 2008).
16. Kanzleiter, R., Improvement in design and delivery of green
beer movement into maturation and the start up and installation
of state-of-the-art centrifuges. Master Brewers Association of
the Americas Convention, Nashville, TN, October 26-28, 2007.
Presentation O-30.
17. Lange, H., Taillandier, P. and Riba, J.P., Effect of high shear
stress on microbial viability. J. Chem. Technol. Biotechnol.,
2001, 76, 501-505.
18. Lentini, A., A review of various methods available for monitor-
ing the physiological status of yeast - Yeast viability and vitality.
Ferment. 1993, 6, 321-327.
19. Lewis, M.J., and Poerwantaro, W.M., Release of haze material
from the cell walls of agitated yeast. J. Am. Soc. Brew. Chem.,
1991, 49, 43-46.
20. Majara, M., O’Conner-Cox, E.S.C and Axcell, B.C., Trehalose-
an osmoprotectant and stress indicator compound in high and
very high gravity brewing. J. Am. Soc. Brew. Chem., 1996, 54,
149-154.
21. Malcorps, P., Haseloars, P., Dupire, S., and Van den Eynde, E.,
Glycogen released by the yeast as a cause of unfilterable haze in
beer. Tech. Q. Master Brew. Assoc. Am., 2001, 38, 95-98.
22. McCourtie, J., and Douglas, L.J., Relationship between cell sur-
face composition of Candida albicans and adherence to acrylic
after growth on different carbon sources. Infect. Immun., 1981,
32, 1234-1241.
23. Ormerod, M.G., Carter, N.P., Meyer, E.W., Loken, M.R., Wells,
D.A., Larsen, J.K., Wilson, G.D., Poot, M., Young, B.D.,
Monard, S., Rabinovitch, P.S., June, C.H., Tanke, H.J., Watson,
J.V., Dive, C. and Workman P., Flow cytometry a practical ap-
proach. 2nd ed., Oxford UP: London, 1994, pp. 1-28.
24. Pratt-Marshall, P., High gravity brewing- an inducer of yeast
stress. Its effect on yeast cellular morphology and physiology.
PhD Thesis. Heriot-Watt University, Edinburgh, 2002.
25. Quain, D.E., Thurston, P.A. and Tubb, R.S., The structural and
storage carbohydrates of Saccharomyces cerevisiae: changes
during fermentation of wort and a role for glycogen catabolism
in lipid biosynthesis. J. Inst. Brew., 1981, 87, 108-111.
26. Quain, D.E., The determination of glycogen in yeast. J. Inst.
Brew., 1981, 87, 289-291.
27. Quain, D.E. and Tubb, R.S., The importance of glycogen in
brewing yeasts. Tech. Q. Master Brew. Assoc. Am., 1982, 19, 29-
33.
28. Quain, D.E. and Tubb, R.S., A rapid and sensitive method for
the determination of glycogen in yeast. J. Inst. Brew., 1983, 89,
38-40.
29. Quilliam, W.R., Yeast handling and subsequent fermentation
performance in a large modern brewery. Proceedings of the 6th
Institute of Brewing Convention (Central and Southern Africa
Section), 1997, pp.61-66.
30. Sall, C.J., Seipp, J.F. and Pringle, A.T., Changes in brewer’s
yeast during storage and the effect of these changes on subse-
quent fermentation performance. J. Am. Soc. Brew. Chem.,
1988, 46, 23-25.
31. Shen, H.Y., Moonjai, N., Verstrepen, K.J. and Delvaux, F.R.,
Impact of attachment immobilization on yeast physiology and
fermentation performance. J. Am. Soc. Brew. Chem., 2003, 61,
79-87.
32. Siebert, K.J., Stenroos, L.E., Reid, D.S. and Grablowski, D.,
Filtration difficulties resulting from damage to yeast during cen-
trifugation. Tech. Q. Master Brew. Assoc. Am., 1987, 24, 1-8.
33. Slaughter, J.C. and Nomura. T., Intracellular glycogen and tre-
halose contents as predictors of yeast viability. Enzyme Microb.
Technol., 1992, 14, 64-67.
34. Souza, N.O. and Panek, A.D., Location of trehalase and treha-
lose in yeast cells. Arch. Biochem. Biophys. 1968, 128, 22-28.
35. Stewart, G.G., The influence of contemporary brewing proce-
dures on yeast handling and condition. Proceedings of the 25th
Institute of Brewing Convention (Asia Pacific Section), 1998,
pp.56-60.
36. Stewart, G.G., Yeast management – The balance between fer-
mentation efficiency and beer quality. Tech. Q. Master Brew.
Assoc. Am., 2001, 38, 47-53.
37. Stewart, G.G. and Russell, I., An introduction to brewing sci-
ence and technology - Series III - Brewer’s Yeast. The Institute
of Brewing: London, 1998, pp. 59, 66-69.
38. Stoupis, T., Stewart, G.G. and Stafford, R.A., Mechanical agita-
tion and rheological considerations of ale yeast slurry. J. Am.
Soc. Brew. Chem., 2002, 60, 58-62.
39. Thevelein, J.M., Regulation of trehalose mobilization in fungi.
Microbiol. Rev., 1984, 48, 42-59.
40. Thomas, C.R., Shear effects on cells in bioreactors. In: Proceed-
ings of Solids-Liquid Suspensions. P. Shamlou, Ed., Butter-
worths Publishing: London, 1993, pp.158-159.
41. Rambeck, W. and Simon, H., Decrease of glycogen and treha-
lose in yeast during starvation and during ethanol formation un-
der the influence of propanol or ethanol. Hoppe-Seylers Z.
Physiol. Chem., 1972, 353, 1107-1110.
42. Wera, S., De Schrijver, E., Geyskens, I., Nwaka, S. and
Thevelein, J.M., Opposite roles of trehalose activity in heat-
shock recovery and heat-shock survival in Saccharomyces
cerevisiae. Biochem. J., 1999, 343, 621-626.
43. Williams, N.J. and Wiseman, A., Loss of yeast flocculence after
release of extracellular invertase (β-fructofuranosidase) and acid
phosphatase by a modified shock procedure that maintains via-
bility. Biochem. Soc. Trans., 1973, 1, 1301-1303.
44. Valli, M., Sauer, M., Branduardi, P., Borth, N. and Porro, D., In-
tracellular pH distribution in Saccharomyces cerevisiae cell
populations analysed by flow Cytometry. Appl. Environ.
Microbiol., 2005, 71, 1515-1521.
45. Van Dijck, P., Colavizza, D., Smet, P. and Thevelein, J.M., Dif-
ferential importance of trehalose in stress resistance in fermen-
ting and nonfermenting Saccharomyces cerevisiae cells. Appl.
Environ. Microbiol., 1995, 61, 109-115.
46. Vitale, M., Zauli, G. and Falcieri, E., Apoptosis vs. Necrosis. In:
Proceedings of the USPHS Workshop 1997. Purdue University
Cytometry Laboratories (CD ROM) 1998.
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