Practical management of yeast: conversion of sugars to ethanol 121

Chapter 10
Practical management of yeast: conversion of sugars to ethanol
DAVE R. KELSALL AND T. PEARSE LYONS
Alltech Inc., Nicholasville, Kentucky, USA
Introduction
Fermentation is the critical step in a distillery. It
is here that yeast converts sugar to ethanol; and
it is here that contaminating microorganisms find
an opportunity to divert the process to lactic acid,
glycerol, acetic acid, etc. The fermentation step
(and to a much lesser degree the cooking
process) is also where yeast must be coached
to produce maximum levels of ethanol,
sometimes as high as 23%. Fermentation will
also have a direct bearing on downstream
processing. If sugar levels remaining in beer are
too high, evaporative capacity may be impaired
or syrup contents increased. Both can lead to
distillers grains being more difficult to dry and
altered in color as Maillard reactions occur
between proteins and sugars. Truly the
fermentation step is the heart of the distillery.
When reflecting on the overall flow diagram
of the distillery (Figure 1) the central role of yeast
is obvious. The process of converting sugars to
ethanol takes between 10 and 60 hrs during
which heat is generated. The various factors
affecting the efficiency of that process need to
be considered. In the chapters by Russell and
Ingledew, the biochemistry of ethanol production
by yeast is covered in detail. In this chapter we
will discuss the factors involved in taking
liquefied mash from cooking (1-2% glucose) to
ensure that pre-formed sugar and other bound
sugars (starch and other polysaccharides) are
released in a timely fashion for maximum
conversion to ethanol by yeast.
Choosing a yeast strain
One of the most important decisions a distiller
must make is the selection of yeast strain. The
best characterized yeast is Saccharomyces
cerevisiae, but within this species there are
thousands of unique strains. Which one to use?
Several decisions must be made (Table 1). The
first decision is whether or not to grow the strain
in-house. In fact, yeast strain maintenance and
growth is a job best left to the experts. For the
very small cost per gallon it represents (less than
0.5 cents), there is just too much at stake.
Table 1. Choices among yeast strains.
Grow in-house?
Active dried or pressed?
High alcohol tolerance needed? Thermosacc
Lower alcohol in fermentor: Superstart
122 D.R. Kelsall and T.P. Lyons
ACTIVE DRY OR PRESSED YEAST?
The choice between active dried (95% solids)
or pressed yeast (35% solids) very often comes
down to two points: adequate cold storage
(pressed yeast must be stored at 5C and has a
shelf life of 1-2 months) and whether the yeast
must start immediately. Yeast requires time to
adapt to any new environment and activate its
metabolism, but first must be rehydrated. This
is a period of zero growth but intense
biochemical activity. If we are conditioning the
yeast, the advantage of a pressed yeast is that
the lag phase is minimized because the yeast is
already hydrated. Cell numbers are typically 7-
10 billion cells/g. Active or instant dry yeast
(ADY) are vacuum packed, often under
nitrogen, and are easy to store with minimal
losses in activity (5% over 6 months at 5C).
ADY requires wetting, and the lag phase tends
to be 1-2 hrs. Cell numbers 15-20 billion cells/g.
IS HIGH TEMPERATURE AND/OR HIGH
ALCOHOL TOLERANCE NEEDED?
Yeast is sensitive to temperature, high or low,
and responds by activating stress mechanisms
1 bushel of
corn
(56 lbs)
Fermentable
sugar
(36 lbs)
Ethanol
(17.6 lbs)
CO2
(18.4 lbs)
Heat
(7450 BTU)
DDGS
(17 lbs)
The distillery flow diagram:
from corn to ethanol plus
co-products
+
+
+
Starch
(32 lbs)
Milling
Liquefaction
Saccharification
Fermentation
Recovery
Dryhouse
Figure 1. Distillery product flow.
including elevation of intracellular trehalose.
Additionally, certain proteins, called heat shock
proteins, are synthesized. Some strains are better
able to withstand temperature stress than others.
Such yeast are also usually able to tolerate higher
alcohol levels. In the 1980s it was customary to
run fermenters at 8-10% ethanol but now 16-
17% is the norm. One yeast, Thermosacc , is
a good example of a stress resistant strain.
Smaller in size (5 m instead of 8-10 m, Figure
2), it has a temperature tolerance of >40C and
an ethanol tolerance in excess of 20%. An
unusual feature is its ability to tolerate high acetic
and lactic acid levels. This may explain
observations of cleaner fermentations as
infecting microorganisms such as Lactobacillus
have less impact. In beverage alcohol
production, little, if any, change in congeners
has been reported following its use.
Controlling yeast stress factors that affect
alcohol yield
TEMPERATURE: THE FIRST STRESS FACTOR
Inability to precisely control the temperature of
Practical management of yeast: conversion of sugars to ethanol 123
fermentation is possibly the biggest and most
commonly encountered factor or problem
affecting alcohol yield. Almost all distilleries
suffer from underestimation of the amount of
energy released during fermentation. Consider
the general conversion of glucose to alcohol as
illustrated in Figure 1. Generation of most of
the energy of fermentation of ethanol takes place
between hours 10 and 30 of fermentation. In
other words, the fermentor cooling system
should be designed to cool 44,000 BTU per 100
lbs of ethanol produced over a 20 hr period.
Often this amount of cooling is not designed
into the system and the fermentors become
overheated. Engineers did not anticipate the
enormous strides that would be made in yeast
fermentation (higher alcohol tolerance, faster
conversion) and designed for shorter residence
times and less cooling capacity.
The theoretical temperature rise for a 25 gallon
beer (25 gallons of water per bushel of grain) is
22
o
C per bushel (Figure 3). The optimum
temperature of fermentation for yeast
(Saccharomyces cerevisiae) is 35C and the
optimum temperature of reproduction for the
yeast is 28C (yeast capable of reproducing at
35C is now available). It can also be seen (in
Figure 3) that without adequate cooling, a
fermentor set at 35
o
C would inevitably increase
in temperature. Every degree in temperature
above 35C will have a tendency to depress
fermentation, as the yeast cannot handle these
higher temperatures. Furthermore, the increase
in temperature favors the growth of lactobacilli,
Figure 2. Thermosaccvs regular yeast.
25 8O 85 4O 45 5O
Mash bill gallons (gallons of water per bushel)
5
1O
15
2O
25
8O
T
e
m
p
e
r
a
t
u
r
e

r
i
s
e

(

C
)
Figure 3. Theoretical temperature increase during
fermentation.
124 D.R. Kelsall and T.P. Lyons
the microorganism that competes with yeast for
glucose. Heat also increases the impact of other
stress factors such as mycotoxins and salt levels.
Combining enzyme activities to control sugar
delivery to yeast
How can we best control fermentation
temperature? The easiest way to reduce
fermentor temperature would be to reduce the
sugar level going in and therefore reduce yeast
growth; however, less alcohol would be
produced. Since the objective is more, not less
alcohol, this is not an option. However, sugar
level, or more importantly sugar type, is
important.
Yeast must be maintained in a growth
(budding) phase, since their ability to produce
alcohol is more than 30 times greater during
growth than in non-growth mode. Yeast however
can respond to high glucose levels in two ways.
Cell growth is either inhibited by high glucose
levels or yeast may grow rapidly and then stop.
In either case there is an over-dosing effect on
fermentation efficiency and possibly a spike in
temperature. Glucose must be spoon-fed to the
yeast; and for this reason it is recommended that
glucoamylases be used both sparingly and in
conjunction with an enzyme produced in surface
culture called Rhizozyme. The combination
provides ideally balanced sugar delivery to
maintain good yeast growth and maximum
alcohol productivity while avoiding high
temperature peaks. Glucoamylase (Allcoholase
II L400) provides glucose at the start of
fermentation while the Rhizozyme, with pH
and temperature optimums more closely aligned
to fermentation conditions, continues to work
during fermentation. The net result is a steady,
slow release of glucose and a steady formation
of alcohol by yeast.
Rhizozyme
TM
also affects ethanol yield as it is
capable of hydrolyzing starch that is
unconverted in the cook process and also
converts some of the cellulose in the corn to
glucose.
The key is to consider cooking and
fermentation together. Cooking must be
designed to release as much starch, both bound
and unbound, as possible without overloading
the yeast with glucose. It must also be recognized
that corn has, in addition to starch, other
potentially useful carbohydrates including
cellulose and hemicellulose. Conventional
glucoamylases do not degrade either, whereas
glucoamylase produced by surface culture
fermentation (Koji) also contains a range of
carbohydrase enzymes as side activities that
degrade some of the cellulose thereby releasing
more sugar, which boosts yield (Table 2,
Figure 4).
Glucoamylase
E
t
h
a
n
o
l


(
%

v
/
v
)
Rhizozyme
O
5
1O
15
2O
25
O.O4 O.OG O.1O
Figure 4. Comparison of alcohol yields from high solids corn mash with traditional and surface culture glucoamylases.
Practical management of yeast: conversion of sugars to ethanol 125
Table 2. Comparison of surface culture and
conventional glucoamylase.
Production Surface culture Conventional
(Koji) fermentation deep tank
liquid
Activities
Glucoamylase 40 400
Cellulase 400
Hemicellulase 350
-amylase 5000
Phytase 300
pH profile Broad Narrow
Temperature
optimum, C 30-40 0-60
Activity on
gelatinized starch Rapid Slow
Activity on raw
starch Reasonable Slow
Yeast stimulation Significant Poor
Modeling provides a prediction of the increase
in yield to be expected. Predicted yields of 3.1
gallons per bushel, 17-18% higher than the
standard 2.65, have been calculated based on
increased sugar release. This is 18 gallons (>65
liters) more per tonne of grain. The
recommendation is to use a 50/50 blend of
Rhizozyme Koji and conventional
glucoamylase to maximize yield. Where distillers
can reliably measure yield, the results in terms
of costs per gallon reveal optimum returns.
Equipment for heat control
Despite the sequential feeding of glucose, heat
is generated during fermentation and must be
removed. Four types of heat exchangers are
typically used; and these are illustrated later in
this chapter when fermentation design is
discussed.
a) Internal cooling coils. These are the least
desirable option because they are practically
impossible to clean and sterilize.
b) Internal cooling panels. Like cooling coils,
panels are suspended in the fermentation
vessel. Also like cooling coils, cooling is
adequate but sterilization may be difficult.
c) External cooling jacket. Cooling jackets,
often dimpled for added contact with the
fermentation, can cover either part of the
vessel or the entire vessel. Recirculating
coolant through the jacket allows
fermentation temperatures to be monitored.
If jackets are spaced out over the fermentor
sides, then cooling can commence as the
fermentor is filled.
d) Heat exchanger with recirculation. Contents
of the fermentor are pumped through a heat
exchanger (typically spiral or plate-and-
frame) and returned to the fermentor. This
has the added advantage of acting as a means
of keeping the fermentor agitated; and
nutrients (sterol donors, peptides, oxygen,
etc.) can be introduced at critical times. It is
suggested that optimum control of
temperature can be obtained by placing the
heat exchangers in such a position that
recirculation can start at the beginning of the
fill when the yeast starts producing heat.
Of the four types, the cooling jacket is best for
minimizing infection, but the heat exchanger is
the most effective at heat control. As the distiller
moves towards very high levels of ethanol (over
20% by volume), heat recovery and maintenance
of low temperatures will become even more
important as temperature rises of 6-8C will
become the norm. A word of caution: some
distilleries, particularly in continuous operations,
share external heat exchangers between
fermentors in order to economize. Based on the
experience of many existing distilleries using this
sharing concept, infection can easily be
transferred among fermentors as well as having
inadequate cooling.
INFECTION: THE SECOND STRESS FACTOR
The various types of infection are described in
detail elsewhere in this book, however several
points are relevant in this context. Lactobacilli
consume glucose to produce lactic acid and
acetic acid, which is the second major factor
affecting the yield of alcohol in fermentation,
which in turn has a major impact on distillery
economics (Table 3). Yeast do produce some
organic acids during fermentation, but
concentrations are relatively low compared to
those produced by lactobacilli and other
contaminating bacteria. As a general rule of
126 D.R. Kelsall and T.P. Lyons
thumb, if the titratable acidity of an uninoculated
mash is X, then the titratable acidity of the
fermented beer is usually about 1.5X. This is a
general rule and seems to work quite well in
practice, although it is not based on known
scientific principles. The 1.5X represents the
titratable acidity of the finished beer when there
is no significant contamination from lactobacilli.
When lactobacilli are active, the generation of
lactic and acetic acids substantially increases the
titratable acidity and often the high acid content
will cause the yeast fermentation to stop or
dramatically slow down. Very early into an
infection the titratable acidity reaches 2X.
Practical means of controlling infection
The most fundamental way to control infection
is to control the fermentation environment (Table
4). Fermentors must be cleaned on a regular
basis; and designs must avoid sharing of heat
exchangers and dead legs in piping. Fermentors
should be designed to facilitate complete
emptying. Scaling will occur regardless of the
fermentor design, and should be removed using
either a scale inhibitor (such as Scale-Ban
TM
) or
a de-scaling chemical (an acid wash).
Fermentations must be fast; and yeast should be
added either in a form that will quickly
rehydrate or preferrably one that has been
started in a conditioning tank. Saccharification
tanks can be a source of infection as
lactobacillus strains can adapt and grow at the
temperatures used in this process. The goal is to
make conditions as suitable as possible for yeast
growth (without jeopardizing alcohol yield) and
as unsuitable as possible for infectious
microorganisms. By restricting glucose release
in fermentation by using Rhizozyme and
lower levels of glucose-releasing glucoamylase
(typically 50-70%), the yeast uses the sugar as
it becomes available and competitively excludes
lactic and acetic acid-producing bacteria by
reducing available sugar. Equally, when yeast
growth rate begins to decline (typically at 20-
24 hrs) sterol donors and oxygen, which at that
stage have become limiting, will revive activity
by ensuring that fatty acids are available for
yeast reproduction.
Table 4. Practical means of controlling infection.
Avoid deadlegs in lines
Avoid sharing fermentor heat exchangers
Proper cleaning programs
Periodic use of de-scaling chemicals
Control fermentation temperatures
Avoid the saccharification step in cooking
Maximize yeast growth (budding)
Use yeast yeast conditioning
Use sterol donors and peptides as yeast foods in yeast
conditioning
Antimicrobials
Allpen
TM
(against Gram-(+) organisms)
Lactoside
TM
(against Gram-(+) and Gram-(-) organisms)
Regardless of precautions, a distillery is not a
sterile environment and opportunities for growth
of both Gram (+) and Gram (-) bacteria exist.
Because their growth rate is so much faster than
yeast, strict controls to prevent infection must
be in place. As early as 1954 penicillin was used
in ethanol fermentation; and owing to its quick
inactivation caused no problems with regulatory
agencies. Other antibiotics such as
virginiamycin are effective antimicrobials, but
some are now known to suppress yeast
fermentation and in addition can leave residues
(2.6%) in spent grains. With the ban in Europe
against use of virginiamycin in products destined
for animal feeds, caution is urged. Furthermore,
Table 3. Relationship between bacterial numbers and ethanol production losses.
Viable bacteria Ethanol lost
1
Ethanol lost
2
Financial loss
3
in mash at set (CFU/ml) (% v/v) (gallons) (in US dollars)
105 0.1 - 0.2 80,000 112,000
106 0.2 - 0.4 160,000 224,000
107 0.6 - 1.0 400,000 560,000
108 0.9 - 1.2 480,000 672,000
109 1 - 1.5 600,000 840,000
1
Depending on the strain of Lactobacillus.
2
Maximum loss calculated based on a 40,000,000 gallon/yr.
3
Calculated based on ethanol price of $1.40/gal.
Practical management of yeast: conversion of sugars to ethanol 127
resistance to both penicillin and virginiamycin
is increasingly common.
One of the strategies to avoid this risk is to
take advantage of the synergy that occurs when
antimicrobial substances are combined, which
lowers the amounts used. Lactoside, a
combination of antimicrobials without
virginiamycin, has proven very effective for this
reason. A routine maintenance dose of 1-2 ppm
is practical as a precautionary measure. When
there is infection the dosage is increased to 2-5
ppm. There is no evidence of carryover of these
antibiotics into spent grains and solubles.
ALCOHOL LEVELS: A THIRD STRESS FACTOR
High alcohol beers have a tendency to stop
fermenting. Understanding how alcohol levels
act to stress yeast requires a working knowledge
of yeast metabolism and growth. It has been
shown that when yeast are in the reproductive
phase they produce alcohol over 30 times faster
than when not reproducing. Figure 5 shows
typical yeast growth patterns or phases during
fermentation. The first phase is the so-called lag
phase, during which the yeast adapt to the
fermentor environment. During this period there
is little or no yeast growth and consequently little
or no alcohol production. This period can last
from 4 to 12 hrs. Ways of managing and
reducing the length of the lag phase are shown
in Figure 6 in reference to yeast conditioning.
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Figure 5. Typical yeast growth curve in distillery mash.
10,000 - 20,000 gallons
Hold for 8 hours
Good agitation
Good cooling
Can be sterilized
Figure 6. Yeast conditioning tanks: A key factor in
maximizing yeast cell numbers and improving alcohol levels.
The second phase is the exponential growth
phase. This is the most important phase where
nearly all of the alcohol is produced. There is a
limited time in which the yeast stay in this phase;
and this is the factor limiting the quantity of
alcohol produced during a given fermentation.
The length of time during which the yeast can
remain reproducing depends on the nutrition
available in the fermentor.
The three major factors that significantly affect
alcohol yield are therefore temperature, acidity
of the mash and the alcohol level produced
during fermentation. Yeast is tremendously
resilient and usually produces efficiently despite
all the negative conditions imposed. Yeast
fermentation will yield well if the temperature is
a few degrees high or if the acidity is a little
high indicating a mild infection. Beers
containing up to 20% alcohol v/v can be
produced without slowing fermentation.
However, when changes in two or more of these
factors happen simultaneously, serious losses in
yield usually occur. For example, in a mash at
37C contaminated with lactobacilli at 10 million
CFU/ml, it is likely that fermentation would stop
at 8% alcohol, a 33% loss in yield.
MAXIMIZE YEAST PERFORMANCE BY
CONDITIONING
Yeast is the powerhouse of any distillery; and
without healthy yeast, alcohol percentages in the
fermentor and alcohol yield will drop.
Conditioning tanks are a way to ensure good
cell numbers in the fermentor (Table 5). At the
start of batch fermentation there should be a
128 D.R. Kelsall and T.P. Lyons
minimum of 50 million cells/ml, which should
increase to 150-200 million cells/ml at the height
of fermentation and drop to 100 million cells/
ml at the end. Continuous fermentors should
maintain cell numbers of 150 million from a high
of 250-300 million cells/ml. Yeast viability as
measured by methylene blue stain should be 98-
99% in the conditioning tank and 90% at the
start of fermentation. There will be a drop to
around 50% viability at the end of fermentation.
In a cascade continuous fermentation system,
viability was seen to drop to 30% in the last
fermentor.
Table 5. Keys to a good yeast conditioning tank (pre-
fermentor).
Keep Brix to 10-20, preferably ~14Brix
Provide good agitation
Use peptide based yeast food
Achieve minimum of 200 ppm FAN (free amino nitrogen)
Add Rhizozyme to spoon-feed yeast glucose
(0.05% of mash)
Hold for 4-8 hrs with agitation
Achieve 300-500 million cells/ml before transfer to main
fermentor
Pump to fermentor
Clean and sterilize
Repeat
A typical charge for a continuous yeast
conditioning tank is shown in Table 6. The mash
was sterilized and cooled to 90F. Yeast was
added with good agitation along with yeast food
and saccharifying enzyme (Rhizozyme). The
mash was held for 8 hrs at which stage a cell
count in excess of 500 million was achieved.
The mash was transferred to the fermentor and
the tank cleaned and sterilized. In this way
distillers ensure good yeast growth with
minimum lag phase periods.
Table 6. Typical charge for yeast conditioning tank for a
500,000 gallon fermentor.
Prefermentor size 20,000 gallons
Mash Direct from jet cooker: dilute to
14Brix
Active yeast 200 lb Thermosacc
TM
Peptide yeast food 20 lbs AYF
Rhizozyme l lb
Yeast cell numbers
expected 400-500 million
Time 8 hrs
MYCOTOXINS: A STRESS FACTOR
In 1985 the Food and Agricultural Organization
(FAO) of the United Nations estimated that at least
25% of the worlds grain supply is contaminated
with mycotoxins. The type and degree of
contamination depend on many factors including
conditions during growth and harvest and
geographic area. Toxins such as zearalenone and
vomitoxin (deoxynivalenol) produced by growth
of Fusarium molds in stored grain tend to be the
mycotoxins of concern in the more temperate
climates of North America and Europe while
aflatoxin, produced by various species of
Aspergillus, predominates in tropical climates and
can be a serious factor in parts of the US. However
a wide range of toxins and mixtures of toxins are
usually present and all are of concern in the animal
feed and human food chain. To a distiller, the main
concern with mycotoxins is whether they pass
through into the DDGS. The FDA have set
definitive limits on aflatoxin levels for interstate
grain shipments; and the increasing knowledge of
the variety of mycotoxins and extent of
contamination possible have heightened concern
in all of the animal feed industry.
Of more recent concern to the distiller has been
the finding that mycotoxins can also affect yeast
growth (Table 7). Perhaps presence of these toxins
may provide at least part of the reason behind
unexplained unfinished fermentations. Since not
all mycotoxins are destroyed by heating, it is
unlikely that the cooking step would remove or
inactivate them. In animals many of the
mycotoxins damage protein metabolism causing
reduced growth and increased disease
susceptibility. Research into ways of nutritionally
compensating for mycotoxin presence in animals
feeds led to discovery that yeast cell wall
glucomannans have characteristics that make them
very specific adsorbents for certain mycotoxins.
For example, about 80% of the zearalenone in
solution can be bound and inactivated by adding
yeast glucomannans. These products are also
beginning to be used successfully in the
fermentation industry.
Table 7. Effects of mycotoxins on yeast growth.
Mycotoxin Level required to inhibit
yeast growth (ppm)
Zearalenone 50
Vomitoxin 100
Fumonisin 10
Practical management of yeast: conversion of sugars to ethanol 129
PHYTIC ACID: AN ANTINUTRITIONAL FACTOR
Phytic acid, the form in which 60-80% of the
phosphorus is stored in cereal grains, is a stress
factor for yeast that may be converted into a
nutritional benefit. The phytic acid molecule has
a high phosphorus content (28.2%) and large
chelating potential. In addition to holding
phosphorus in tightly bound form, phytic acid
can form a wide variety of insoluble salts with
di- and trivalent cations including calcium, zinc,
copper, cobalt, manganese, iron and
magnesium. This binding potentially renders
these minerals unavailable in biological systems;
and makes phytin content a familiar
antinutritional factor when cereal grains are fed
to livestock. Animal nutritionists either
compensate for the presence of phytin with
extra-nutritional levels of zinc, calcium and
phosphorus or add microbial phytases to diets
for pigs and poultry.
Phytin may also have an antinutritional effect
on yeast. Calcium ion concentration affects
enzyme activity; and minerals such as zinc are
needed for yeast growth. Phytic acid is also
known to inhibit proteolytic enzymes; and
phytate-protein or phytate-mineral-protein
complexes may reduce the breakdown of protein
to amino acids required by yeasts for growth.
Starch is also known to be complexed by phytate.
Finally, inositol, a limiting nutrient for yeast
growth, forms a part of the phytin molecule. The
addition of a phytase enzyme (often present
naturally in Koji surface culture enzyme sources)
can overcome this problem.
SUMMARY: STRESS FACTORS AFFECTING
YEAST ACTIVITY
As we coach yeast to produce very high levels
of ethanol, it becomes of paramount importance
that we both understand the stress factors
involved and design equipment to allow us to
control them (Figure 7). Critical stress factors
such as temperature, alcohol levels and infection,
while understood individually, combine to
produce unique conditions in every
fermentation. Such conditions will vary with
feedstocks used, but also with changes in the
composition of individual feedstocks due to
seasonal or climatic variables. An understanding
Figure 7. Stress factors affecting yeast.
130 D.R. Kelsall and T.P. Lyons
of other feedstock-related stress factors such as
mycotoxins or phytin content will also become
more important as we move toward higher
ethanol production.
Fermentation systems used in distilleries
Before considering the specific management
tools available to the distiller, it is necessary to
consider the differences among the fermentation
systems in use. Over the last 30 years the
brewing and distilling industries have developed
new fermenting systems; and the distilling
industry has largely converted to rapid batch
fermentation with cylindro-conical or sloping
bottom fermentors, or to cascade continuous
fermentation using cylindro-conical fermentors.
BATCH FERMENTATION
A typical cylindro-conical fermentor with skirt
support is illustrated in Figure 8. These
fermentors are usually designed to ferment from
30,000 gallons to 750,000 gallons. Fermentors
need to be fabricated on site if the diameter and
length exceed a size that can be safely
transported by road.
Figure 8. Alcohol fermentor with sloping bottom (Alltech/Bishopric, 1981).
Inspection light
Vent
From CIP module
Mash in
Yeast/enzymes in
20 in. Manway/
sampling port
Coolant
Insulation
(outdoor tanks
in cold climates)
Slope
Concrete pad
Alternative cooling methods:
A. External cooling jacket
B. External heat exchanger with recirculation
C. Internal cooling coils
D. Internal cooling panels
CIP spray head
A
15 in. x 20 in.
access Manway
Recirculation pump
Outlet
B
C
Agitator
(if required)
D
Coolant
Practical management of yeast: conversion of sugars to ethanol 131
Different cooling systems are available. Many
distilleries use chilled water to cool the
fermentors. From a microbiological standpoint
the most desirable cooling system by far uses
external cooling jackets. The least desirable
option employs internal cooling coils because
they are practically impossible to clean and
sterilize from the top-mounted CIP (clean-in-
place) spray nozzles. The external recirculation
heat exchanger is frequently used in distilleries
and is sometimes shared with other fermentors.
This is a poor option as there are times when
more than one fermentor needs to be cooled.
The main disadvantage of this system when
shared among fermentors is bacterial cross-
contamination. With such a system, the
microbiological condition of all fermentors will
be determined by the fermentor with the most
contamination. Even when these recirculating
heat exchangers are not shared, they are difficult
to clean. Throughput of at least 7 ft
3
/sec must
be obtained to ensure turbulent flow through the
tubes.
The agitator is required particularly at the start
and at the end of fermentation. Frequently the
agitators are badly designed and provide poor
mixing. A folding action is required to ensure
proper mixing of the mash solids and to ensure
an even temperature throughout the fermentor.
Carbon dioxide (CO
2
) is removed through the
vent. Fermentors must be fitted with pressure
relief valves and vacuum breakers to avoid
serious accidents. The CO
2
is frequently
collected and sold. In any case, CO
2
should be
scrubbed to remove alcohol, which is returned
to the beer well. The CO
2
header manifold can
be a source of contamination as infected mash
from one fermentor can migrate in the CO
2
into
another non-contaminated fermentor. The CO
2
manifold should be designed to be cleanable.
The lag phase is a great opportunity for
lactobacilli to become established. Bacteria
under optimum growth conditions can reproduce
every 20-30 minutes; however yeast, which are
much larger microorganisms, can only
reproduce every 3 hrs. A single bacterium
reproducing every 20 minutes would produce a
population of 256 in 3 hrs. Some yeast strains
have lag phases as long as 12 hrs, during which
time the lactobacilli can become heavily
established. Therefore, it is very important to
choose a yeast strain with a very short lag phase
and to use a conditioning tank so that the main
fermentation starts immediately after the yeast
is added.
There is a choice of materials to use in the
manufacture of fermentors. Stainless steel is
generally the best choice as it is easier to clean
and sterilize. It also lasts much longer than mild
steel or lined vessels; and when considered over
the expected lifetime of a fermentor it is by far
the most economical option. If chloride levels
are high, then special stainless steels should be
considered to avoid stress corrosion.
It is important that the slope of the bottom be
sufficient for the mash to run out when
discharging. This type of fermentor is much
better suited for use with clear or semi-clear
mashes than with whole cereal mashes.
Fermentor cleaning is accomplished with
clean-in-place (CIP) equipment. CIP sprayheads
are high-pressure devices (100-120 psi), which
usually have automated cleaning cycles. A
typical cleaning cycle would be (minimum):
Pre-rinse with water 10 minutes
Detergent circulation 20 minutes
Post-rinse with water 10 minutes
Sterilization 10 minutes
The detergent used would be based on caustic
soda, normally with added wetting agent,
antifoam and de-scaling agent. The caustic
strength should normally be in the 3-5% range.
Ideally, the detergent should be hot (80-90C).
The detergent and rinse waters should be
continually drawn off from the fermentor to
prevent accumulation of liquid during the cycle.
The detergent and post-rinse should at least be
recirculated and continually made up to strength.
Chlorine dioxide and iodophors make ideal
sterilizing agents, but many distillers still use
steam to sterilize the fermentors. This is time-
consuming and is probably not as effective as
chemical sterilization.
It is important to control the build up of
beerstone (calcium magnesium phosphate and
calcium oxalate). Bacteria can penetrate
beerstone, which is insoluble in straight alkaline
solutions. Beerstone thus protects the bacteria
from detergent and sterilant. EDTA-based
chelating agents added to the detergent help
dissolve beerstone and will control
132 D.R. Kelsall and T.P. Lyons
accumulation. The level of chelating agent
needed can be calculated from the calcium level
in the mash. A problem recently encountered is
sodium contamination of process condensate
water. During CIP the caustic based detergent
can be neutralized by the CO
2
in the fermentor
producing large quantities of soluble sodium
carbonate that can pollute the process
condensate. Very high sodium levels can weaken
the yeast during fermentation and care must be
taken to reduce these sodium levels.
CONTINUOUS FERMENTATION
The most successful continuous fermentation
system used in distilling is the cascade system
(Figure 9). The system illustrated in Figure 9 has
at least two fermentors and a beer well where
the yeast are recycled. Yeast can only be recycled
when clear mashes are used. The yeast can then
be centrifuged, washed and reused. Typically,
yeast can be washed using phosphoric acid at a
pH of 2.2-2.4 and a holding time of 90 minutes.
These conditions are sufficient to kill the bacteria
without doing permanent damage to the yeast.
Chlorine dioxide at 40-50 ppm is an alternative
to acid washing, and seems to be gentler on the
yeast.
The system in Figure 9 has two fermentors,
whereas most cascade plants have five
fermentors and a pre-fermentor. The majority of
these plants in the US use whole mash fed into
the first two fermentors. Both are aerated
continuously with sterile air and are cooled with
external coolers. The pre-fermentor feeds yeast
equally into both. The mash has been previously
saccharified and enters the fermentors at a pH
of 4.0 or slightly less. Usually these systems, if
yeast stress factors are taken into account,
produce 13-15% beers in 24-30 hrs although
clear mash systems operate in 10-14 hrs. Each
fermentor is individually cooled and agitated
either with air or CO
2
or mechanically. The clear
mash systems are capable of maintaining a fixed
yeast count although they have the potential to
recycle.
Typically yeast cell counts in these fermentors
are slightly higher than in batch systems, with
counts in the range of 180-220 million cells/ml
for the first two fermentors and slightly less as
the mash proceeds through the system. The
viability and percentage of budding cells
decrease from one fermentor to the next.
Typically, the beer entering the beer well could
be down to 120 million cells/ml with a viability
of around 30%. Alcohol levels could be 10-11%
in Fermentors 1 and 2, 12-13% in Fermentor 3
and up to 15% in Fermentors 4 and 5.
The main advantages of continuous
fermentation (and they are very important
advantages) are rapid throughput and the fact
Figure 9. Twin vessel continuous stirred fermentor.
Vessel
No. 2
Vessel
No. 1
Agitator drive
Oxygen
column
Oxygen in
Sterilizer
Mash in
Pump
Agitator drive
Foam breaker
Beer
outlet
Yeast
outlet
Cooling
Coil
CO
2
outlet
Practical management of yeast: conversion of sugars to ethanol 133
that these fermentors can be run for very long
periods without stoppage. Cleaning costs are
therefore practically eliminated; and vessel
utilization is superior to any batch system. These
continuous systems are frequently used for 12
months without stopping and are only cleaned
when the plant has its annual shutdown.
The most serious problem encountered with
the cascade system is infection. The fermentors
can be infected through non-sterile air injected
into the first two fermentors. In this case, acetic
acid bacteria, which convert the ethanol to acetic
acid, can be introduced. This can be detected
by a vinegar odor, or more accurately by HPLC
analysis. There is also a possibility of infection
by lactobacilli that usually propagate in
contaminated mash coolers or saccharification
tanks.
The new distillers, at least those in the US, have
elected not to use continuous fermentation and
are instead using the batch process. Batch
systems are more easily controlled, more flexible
and capable of higher ethanol levels.
Conclusions
Understanding the stress factors that affect yeast
and the fermentation equipment will help us
move to the theoretical 23% ethanol that yeast
such as Thermosacc are capable of producing.
It is critical, however, that we push the positives:
spoon-feed sugar to yeast, provide yeast with
peptides to ensure high cell numbers in early
stages of fermentation, control temperature and
overcome the negatives. These negatives,
infection, lactic acid, acetic acid, phytic acid and
mycotoxins all prevent yeast, and therefore the
distillery, from achieving potential yields.
Today distilleries must achieve yields of 2.9
gallons per bushel. Tomorrows distilleries will
need over 3.2 gallons per bushel. To do this,
yeast must remain the central focus of all efforts.
Doing so will allow 23% ethanol to soon become
the norm.
Continuous fermentation in the fuel alcohol industry: how does technology affect yeast? 135
Chapter 11
Continuous fermentation in the fuel alcohol industry: How does the
technology affect yeast?
W.M. INGLEDEW
Applied Microbiology and Food Science Department, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada
Introduction
Fuel alcohol manufacture in North America is
conducted in approximately 70 (soon to be 78)
production plants. The annual output from North
American fuel alcohol distilleries will soon
exceed 3 billion US gallons (11 billion liters).
Both continuous and batch fermentation
technologies are used, with the breakdown
between plants (Table 1) and on a volume basis
(Figure 1) not exactly illustrating the fact that
older and larger wet mills predominantly use
continuous technology whereas the newer and
smaller dry mills predominantly use batch
fermentation.
Table 1. Fuel alcohol plants using continuous or batch
fermentation (2003).
Numbers of US plants (70 total)
Volume Continuous Batch
<11 million gal/year 3 15
12-39 million gal/year 9 15
>40 million gal/year 14 14
In this article, continuous fermentation, the
prolonging of the logarithmic phase of yeast











B
i
l
l
i
o
n
g
a
l
l
o
n
s
Continuous Batch
Figure 1. Volumes of alcohol made in 2003 in 70 US plants by batch and continuous technology.