raw-materials-selection-and-medium-development-for-industrial-fe

Raw Materials Selection and Medium Development
for Industrial Fermentation Processes
SAMUN K. DAHOD, RANDOLPH GREASHAM, AND MAX KENNEDY
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46.1.
INTRODUCTION
This chapter focuses primarily on raw materials and
medium development for microbial fermentation processes.
Although general principles also apply to it, mammalian
cell culture will not be emphasized. This chapter is not
intended to provide a literature search or a review of specific medium types used in specific fermentation processes.
Rather, it is designed to provide practicing microbiologists
and biochemical engineers with a rational basis for medium
development and improvement. At the start of the chapter,
chemically defined fermentation media are considered.
Then, various commercially available ingredients for key
nutrient components of traditional complex fermentation
media are described in generic terms. This discussion is
followed by a discussion of general considerations and a set
of guidelines for medium development and improvement.
The information provided is derived from experiences in
the fermentation industry, and little effort has been made
to cite references for specific examples and dicta mentioned
in the chapter, even though similar information may also be
presented in the literature.
The fermentation medium forms the environment in which
the fermentation microorganisms live, reproduce, and carry
out their specific metabolic reactions to produce useful
products. The importance of this environment cannot be
overemphasized when it comes to the development of a
productive fermentation process. Over the years, substantial
progress has been made in developing fermentation medium
design as a systematic science. However, experienced industrial microbiologists and biochemical engineers will be the
first to point out that this field is as much an art as it is a science. In most industrial fermentations, where the product
is something other than the cell mass itself, there are two
distinct biological requirements for medium design. First,
nutrients have to be supplied to establish the growth of the
organism. Second, after growth is established, proper nutritional conditions have to be provided to maximize product
formation. Besides these obvious biological requirements,
one needs to worry about selection of nutrient components
that are cost-effective, readily available, and consistent
from lot to lot. In recent years, as integrated approaches to
fermentation and downstream processing have been developed, it has also been recognized that the fermentation medium should not unduly hinder the downstream processing
and, if possible, should even facilitate downstream processing. For new fermentation processes brought up from microbiology laboratories, considerable flexibility and latitude
in medium design are possible. The process is not locked
into a fixed set of raw materials (for example, due to a Food
and Drug Administration [FDA] filing), and the medium
components can be freely selected for the sole purpose of
maximizing the product yield and minimizing the cost. For
an established fermentation process, the choice of medium
components may be limited by such factors as FDA filing,
the cost structure for the product, and the requirements of
downstream processing. In spite of these limitations, continued medium development remains a necessity so that
an established product retains its competitive edge in the
marketplace.
While literature reports for medium development in
specific fermentation processes are plentiful, a general treatment of broad principles involved in fermentation medium
development is comparatively harder to find. Readers may
find the reviews by Kennedy and Krouse (9) and Corbett
(4) informative.
46.2. CHEMICALLY DEFINED
FERMENTATION MEDIA
Chemically defined media have been used routinely in
the laboratory to study the microbial biosynthesis of
primary and secondary metabolites. A key characteristic
that has made these media desirable is consistency of
performance. Although chemically defined media may be
used to study the biosynthesis of the metabolites, complex media are usually used to produce them. Today, most
fermentation processes employ complex media. However,
chemically defined media are becoming popular where
process consistency is very important, such as the production of biologics. Frequently, the process for making the
biologic is considered part of the product definition. In
addition to enhancing process consistency, chemically
defined media have several other favorable characteristics, including better control and monitoring of the fermentation process, rapid scale-up, reduced sensitivity to
large-scale sterilization conditions, and simplification of
downstream processing. Concerns associated with these
media include medium cost and development time as
well as cell growth and production levels. Some of these
concerns are being favorably addressed by the availability
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biological engineering and scale-up of industrial processes
of more-sophisticated analytical tools to quickly analyze
initial complex medium components, the use of statistical design of experiments to rapidly develop and optimize
media, and the use of the well-established technique of
nutrient feeding to circumvent nutrient toxicity at high
concentrations and to enhance productivity.
There are several approaches one may employ to develop chemically defined media. One is to perform a thorough search of the literature for chemically defined media
that support growth of the microorganism of choice or a
closely related one. Once identified, it may be optimized
using statistical design of experiments. Another approach
is to simulate complex medium ingredients using modern
analytical tools. For example, in the medium supporting
biotin production, histidine and methionine were found
to replace the complex component casein hydrolysate. In
general, monosodium glutamate and ammonium sulfate
have proven to be good replacements for complex nitrogen
medium ingredients. A third approach is to develop the
initial medium composition based on the typical elemental formula of the cell being C6H11NO3 with a molecular
weight of 161 including ash (the cell dry matter is 90%
organic and 10% ash) (18) and the elemental composition
of the cell as presented in Table 1 (20).
For example, to estimate the amount of glucose (carbon
source) and ammonium sulfate (nitrogen source) required
to support a yeast dry cell weight (DCW) of 100 g/liter,
the following calculations may be used. For the amount
of glucose (for cell growth): (100 g of DCW/liter)(0.48 g
of C/g of DCW)[(180 g of glucose/mol of glucose)/(72 g
of C/mol of glucose)] 5 120 g of glucose/liter. However,
since glucose is also an energy source for cell maintenance,
the total amount of glucose required for both growth and
maintenance is estimated as follows: cell yield 5 grams of
DCW/gram of glucose consumed 5 0.51 (17). Thus, for 100
g of DCW/liter, 100/0.51 5 196 g of glucose per liter. For
the amount of nitrogen: (100 g of DCW/liter)(0.075 g of N/
g of DCW){[132 g of (NH4)2SO4/mol of (NH4)2SO4]/[28 g
of N/mol of (NH4)2SO4]} 5 35.4 g of (NH4)2SO4 per liter.
Similar calculations are performed for the remaining
elements of the microbe. If a growth factor required by the
microbe is known (e.g., yeasts require biotin), it should be
added. If the requirement for growth factors is unknown,
a small amount of yeast extract (0.001 to 0.05%) may be
added initially.
Care must be taken when using this approach to calculate the initial concentration of medium ingredients,
since they may be growth inhibitory. For example, glucose at a concentration of 50 g/liter inhibits the growth
of Escherichia coli and can be detrimental even at much
lower concentrations. To circumvent this inhibition and
achieve high cell densities, appropriate glucose feeding is
usually employed. For example, a chemically defined medium used to produce recombinant human interferon-a1
by E. coli is presented in Table 2 (19). As shown, glucose
was present initially at a noninhibitory concentration
of 30 g/liter. Subsequent glucose feeding was computer
controlled, maintaining glucose below its inhibitory concentration throughout most (14 days) of the 16-day fermentation cycle.
By calculating the cell mass supported by each of the
elements listed in the medium in Table 2 (such as carbon,
nitrogen, phosphorus, sulfur, etc.), ammonium sulfate was
identified as the growth-limiting nutrient. Based on the
nitrogen level, 34 g of ammonium sulfate was calculated to
support a maximum cell mass of 58 g of DCW per liter—the
cell density achieved with the actual fermentation process.
In addition to the production of biologics, chemically
defined media are proving to be profitable for producing secondary metabolites at manufacturing scale; a good example is
the production of penicillin. Examples of chemically defined
media that have proven to be economically successful for
secondary metabolites are presented in Table 3 (8, 21).
When the chemically defined medium was compared
with the initial, optimized complex medium for producing
the secondary metabolite by Streptomyces, the chemically
defined medium reduced the medium cost by 4.5-fold and
increased the titer by 80% at the 800-liter scale.
Typical elemental composition of microbesa
TABLE 1
Element
Composition (% of DCW) in:
Bacteria
Carbon
48
Nitrogen
Yeast
48
Fungi
48
12.5
7.5
6
Phosphorus
2.5
1.7
2.5
Sulfur
0.6
0.13
0.3
Potassium
2.8
2.5
1.4
Magnesium
0.3
0.3
0.2
Sodium
0.8
0.06
0.26
Calcium
0.56
0.2
0.75
Iron
0.11
0.26
0.15
Copper
0.02
0.006
Manganese
0.006
Molybdenum
0.004
0.0002
a
Adapted from reference 20.
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46. raw Materials selection and Medium development
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Production of recombinant human interferon-a1 by E. colia
TABLE 2
Component
Initial medium
KH2PO4
3.0 g/liter
K2HPO4
5.0 g/liter
Feeding solution
(NH4)2SO4
4.0 g/liter
30.0 g/liter
MgSO4∙7H2O
2.0 g/liter
5.0 g/liter
Vitamin B1
0.1 g/liter
2.0 g/liter
Trace metal
3.5 ml
Glucose
30.0 g/liter
Antifoam
0.5 g/liter
pH
7.0
500 g/liter
a
Adapted from reference 19.
46.3. COMPONENTS OF INDUSTRIAL
FERMENTATION MEDIA
As noted above, most industrial fermentation media are
complex formulations containing poorly defined ingredients. Often these ingredients contain multiple nutrients for
the growth of fermentation microorganisms. However, for
the purposes of medium development, a given ingredient is
thought to provide primarily a single nutrient. For example,
soy flour is used primarily to supply complex nitrogen or
protein for the growth of a microorganism. However, soy
flour also contains substantial amounts of metabolizable
carbohydrate and minerals. In the discussion below, the
medium ingredients are classified according to their primary role in the fermentation process. On this basis, we
can classify the fermentation raw materials in four broad
nutrient categories: materials used primarily as sources of
carbon, nitrogen, or minerals, and materials used for special
purposes.
Chemically defined media for representatives of actinomycetes and filamentous fungi
TABLE 3
Medium component
Composition for production in:
Streptomyces
Gliocladium
(A) Medium
Glucose
112.5 g/liter
Sucrose
(NH4)2SO4
170.0 g/liter
16.5 g/liter
8.9 g/liter
K2HPO4
1.5 g/liter
1.73 g/liter
Monosodium glutamate
7.5 g/liter
CaCl2
1.0 g/liter
66.4 g/liter
CaCO3
1.73 g/liter
MgSO4∙7H2O
1.12 g/liter
Biotin
0.17 g/liter
Salt solution (see part B)
20 ml/liter
2.6 ml/liter
P-2000
2 ml/liter
1.7 ml/liter
(B) Salt solution
MgSO4∙7H2O
28.9 g/liter
ZnSO4∙7H2O
0.5 g/liter
5.0 g/liter
CuSO4∙5H2O
0.05 g/liter
0.5 g/liter
FeSO4∙7H2O
0.5 g/liter
5.0 g/liter
MnSO4∙H2O
0.1 g/liter
1.0 g/liter
CoCl2∙6H2O
0.04 g/liter
0.4 g/liter
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46.3.1.
biological engineering and scale-up of industrial processes
Carbon Sources
46.3.1.1. carbohydrates
Glucose is the most frequently used carbohydrate in the
fermentation industry. In the United States, it is derived
from the corn-processing industry. Two types of products
are in use, dextrose monohydrate and hydrolyzed corn syrups containing glucose at a level greater than 95% (called
DE95 or dextrose equivalent of 95%). While dextrose
monohydrate comes in the form of easy-to-handle crystalline material, it is more expensive. This material is used
primarily in small-scale applications as in seed fermentors
and when consistency is of the utmost importance. For the
bulk of the glucose needs, such as for large-scale fermentations and for in-process feeding, the hydrolysate is the more
economical material. If the fermentation microorganism
is able to hydrolyze low-molecular-weight saccharides, less
expensive corn syrups of various lower degrees of hydrolysis
can be used. Industrial fermentation processes such as those
for the production of penicillin can readily utilize hydrolysates with a dextrose equivalent as low as 20 (DE20). In
fact, some processes give higher yields with these highermolecular-weight saccharides than they do with pure glucose. The next level of complexity in these glucose-based
carbohydrates comes in the form of various dextrins. These
are primarily cornstarch products with just enough hydrolysis carried out to make them soluble in the fermentation
medium. The dextrins, cornstarch, other starches (such as
potato starch), and solid substrates in general are rarely used
for in-process feeding. They are generally used as batchedin carbon sources for initial growth of the organism or as
carbon sources that are gradually assimilated by the microorganism during the product synthesis phase. In the United
States, the crudest and the cheapest source of complex
carbohydrate is corn flour. This product is primarily starch
but also contains about 5% protein. An important cost
reduction strategy used by many fermentation companies
is to use crude starch or corn flour in the batch along with
the commercially available enzyme amylase. The amylase
breaks down starch molecules to generate more readily
utilizable carbohydrates. In many fermentation processes,
the primary carbon source (the most readily utilizable, such
as glucose) is metabolized rapidly during the growth phase
and a secondary carbon source (such as oils) is utilized during production.
Sucrose is often used in fermentation processes. In its
crystalline form, sucrose is available as table sugar of various degrees of refinement. The white crystalline sucrose is
generally used in small-scale applications and in seed fermentors. However, it can also be used as a gradually utilized
carbon source in some fermentations in which the organism
has a limited ability for metabolizing sucrose. The use of disaccharides is often explored as alternate carbon sources for
monosaccharides (such as glucose) when catabolite repression is encountered. The crudest form of sucrose comes as
molasses, which contains anywhere from 3 to 10% protein.
In some fermentations (for example, glutamic acid fermentation), this product gives excellent results as a combined
carbon-nitrogen feed.
In the early days of penicillin fermentations, the carbon
source of choice was lactose. This sugar is gradually metabolized by the penicillin-producing organism and hence
can be batched into the medium from the beginning of the
process. However, since the advent of controlled feeding
of glucose, the importance of lactose in the fermentation
industry has decreased. Lactose is available in granular
form for small-scale applications, and it is still used in
some fermentations, especially in Europe, where it is more
readily available than dextrose and corn syrups. The most
economical source of lactose is derived from the cheese industry by-product cheese whey. This product is available in
a spray-dried form and is an excellent source of protein and
minerals besides being a source of lactose.
Other sugars that are used less frequently in the fermentation industry include maltose, mannitol, sorbitol, and xylose. All of these are generally used in their purified forms.
A related carbon source for the fermentation industry is
glycerol. It is useful in many processes as a gradually metabolized carbon source. Additionally, organic acids, such
as acetic acid, may be used on rare occasions as combination pH control agents and carbon nutrients. Minoda (13)
has reported on the potential uses of other unusual carbon
sources for amino acid fermentations.
46.3.1.2.
oils
Various oils are widely used as carbon sources in the fermentation industry, especially in antibiotic fermentations. Oils
can supply both the energy and the growth carbon needs
of the organism. In many antibiotic fermentations, where
the antibiotic backbone is synthesized from low-molecular-weight fatty acids, the oils make ideal carbon sources
since they gradually supply these fatty acids during the
fermentation process. The oils are used both as batched-in
ingredients and as continuous feeds. In some fermentations,
oils play an important auxiliary role even when they are
not actively metabolized by the fermentation microorganism. The yield-enhancing effect of the oil when it is not
metabolized is not well understood. However, oxygen diffusivity in oils is higher than in water, which may have a
beneficial effect on oxygen transfer from bubbles. It is possible that the oil provides protection to cells from excessive
shear forces or that it makes a key micronutrient from the
complex medium more available to the microorganism in
the form of micelles. In fermentations in which oils can be
utilized as carbon feeds, they offer important benefits. First,
the caloric content and the corresponding energy availability per unit volume of feed are appreciably higher for oils
than for carbohydrates. One liter of vegetable oil has more
than twice the utilizable energy as 1 liter of a 55% solution
of glucose. This high energy density allows for lower feed
rates and smaller feed vessels. Consequently, the fermentor
volume management for long-cycle fermentations is easier
with oil-fed fermentations than with sugar-fed fermentations. This enhanced management is true not only because
less feed is introduced into the fermentor but also because
the metabolism of oil does not produce as much water as the
metabolism of sugars. The antifoaming property of the oils
is also beneficial for most fermentation processes. Before
the advent of synthetic defoamers, oils were used for foam
control in many fermentation processes even when the
carbon source of choice was a sugar. However, the oil added
for foam control is metabolized by the organism, and continuous addition is required to control foam. The synthetic
defoamers are more effective because they are not readily
degraded by the fermenting microorganism and they are
cost-effective. In special cases, when the presence of synthetic defoamer interferes with the downstream processing,
oils are still used as defoamers. The antifoaming properties
of several natural oils are reviewed by Vardar-Sukan (16).
The most important oil in the U.S. fermentation industry is soybean oil. It is abundant and relatively inexpensive.
Other oils that are often used are lard oil, fish oil, and oils
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46. raw Materials selection and Medium development
of other plants such as corn, cottonseed, peanut, sunflower,
and safflower. One specialty oil product that is synthetically made and has found application in the fermentation
industry is methyl oleate. Methyl oleate is often used as a
supplemental feed in conjunction with another feed such
as soybean oil. The fatty acid contents of various oils vary
according to their source, and there may be a theoretical
basis for one type of oil to perform better than another
type. However, the choice of oil in a given fermentation is
generally determined empirically. The oil that is used in the
shake flask fermentations during screening of the producing strains very often also gives better results in large-scale
fermentations.
46.3.2.
Sources of Organic Nitrogen or Protein
There are principally three classes of raw materials available
to supply the organic nitrogen or protein requirement of a
fermentation process: (i) those derived from agricultural
products, (ii) those derived from brewery industry by-products, and (iii) those derived from meat and fish by-products.
All of these products supply other important fermentation
nutrients in addition to organic nitrogen.
46.3.2.1. nitrogen sources derived from
agricultural products
The sources derived from agricultural products are the
workhorse ingredients of the fermentation industry. They
include the products of commodities such as various grains
and soybean. The soybean flours, meals, and grits head
the list of applications in antibiotic fermentations. The
popularity of the soy products is based on the fact that
after the soy oil is extracted from the soybeans, the residue
is about 50% protein, which is readily available for cell
growth. In addition, soy flour, meals, and grits contain up
to 30% utilizable carbohydrates. Most minerals required for
microbial growth are also present in soy-based products.
In many seed medium applications, where growth is the
primary consideration, all that is required in the medium
is soy flour along with salts such as magnesium sulfate and
potassium phosphate. A product that is processed very
similarly to soy flour is cottonseed flour. The protein in the
cottonseed flour is less readily available and thus makes a
good slow-releasing nitrogen source. Corn gluten meal is
another readily available product that is suitable as a slowreleasing nitrogen source. Corn steep liquor, a by-product
of the corn milling industry, was very extensively used in
the early years of the antibiotic fermentation industry. In
recent years, though, due to the variability in the product
quality, the liquid form of corn steep liquor has fallen out
of favor. Spray-dried corn steep liquor is now available and
is used in many antibiotic fermentations because it is less
variable. Other agricultural commodities used as nitrogen
sources in the fermentation industry include peanut meal,
linseed meal, wheat flour, barley meal, and rice meal. Plant
or animal hydrolysates produced by the degrading action of
enzymes, usually proteases, are widely used. Should a plant
source be required, soybean hydrolysates can be used, or if
an animal source is acceptable, then casein or whey protein
hydrolysates may be used.
46.3.2.2. nitrogen sources derived from brewery
industry by-products
The brewing industry is an important source of fermentation raw materials. The principal product is the yeast left
over after beer fermentation. The suitability of the yeast
by-product for a given fermentation depends upon the
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method of drying. The yeast may be drum dried or spray
dried. It is also sold as a paste produced by water evaporation in an industrial evaporator. All of these products have
found applications in the fermentation industry as sources
of nitrogen. However, the yeast is never used as the primary
source of nitrogen. Instead, it is thought of as a nitrogen
supplement with additional beneficial nutrients that are not
available from grain-based nitrogen sources. Generally, these
additional nutrients are organic phosphorus and unknown
micronutrients. Brewery yeast is also refined into yeast extracts of different water solubilities, which are more expensive and used in smaller quantities. Yeast extract is often the
single undefined component used in so-called semidefined
fermentation media to provide micronutrients. The brewing
and distilling industries supply two other by-products that
are sometimes used in the fermentation industry: distillers’
solubles, in the form of a concentrate or spray-dried powder,
and leftover grains from the brewing process.
46.3.2.3. nitrogen sources derived from Meat and
fish by-products
Meat and fish products are very rich in protein. So are the
by-products of these industries. The primary meat-based
product is generically known as spray-dried lard water. This
is a by-product of lard processing. The animal bones and
tissues are boiled in water, sometimes in the presence of
proteases, to free the fat. The resulting liquor is separated
into fat and water layers. The water part is rich in proteins
and peptides. This water, when spray dried, gives a product
with a protein content of 80% or greater. The lard water
can be obtained with different degrees of chemical or enzymatic hydrolysis. Hydrolyzed lard water products are sold as
meat peptones under various brand names. A parallel line of
products labeled fish meals and fish hydrolysates is derived
from heat and enzymatic treatment of fish wastes. These
products are generally about 70% protein.
46.3.3.
Minerals
Minerals are used in fermentation media to serve many
purposes, e.g., as major nutrients, as trace metal suppliers,
as ionic strength-balancing agents, as precursors for secondary-metabolite synthesis, as buffering agents, as pH control
agents, and as reactants to remove specific inhibitory nutrients from the medium. The nitrogen-containing salts (e.g.,
ammonium sulfate, ammonium nitrate, sodium nitrate, and
potassium nitrate) can provide a substantial portion of the
nitrogen requirement for cell growth when combined with
organic nitrogen. When salts are used as nitrogen nutrients,
their metabolism invariably results in pH changes in the
medium. For example, when ammonium sulfate is utilized
by the organism, the pH tends to fall, and when sodium
nitrate is utilized, the pH tends to rise. Therefore, it is very
important that adequate buffering or pH control be provided to counterbalance these pH effects. Ammonia used
for pH control has the advantage of regulating pH while
replenishing ammonium nitrogen used up from ammonium
sulfate in the medium.
Another major nutrient supplied as inorganic salt is
phosphorus in the form of phosphate salts. Phosphorus from
soluble phosphate salts is more readily available to the organism than the phosphorus derived from organic nutrients
such as yeast. As a result, it is possible to control the rate of
growth by balancing organic phosphorus against inorganic
phosphorus salts.
Although most organic nitrogen sources such as grain
meals and yeast extracts contain many of the minerals
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biological engineering and scale-up of industrial processes
required for growth, the fermentation medium is often
supplemented with salts that provide elements that are
required in greater than trace quantities. For example,
magnesium and potassium salts and the salts containing
sulfate are generally included in the medium if they have
not already been included for other purposes. Trace elements such as iron, zinc, manganese, copper, cobalt, and
molybdenum are generally not included in fermentation
media containing high concentrations of complex ingredients unless they serve specific purposes in metabolism. For
example, if product synthesis is known to be carried out by
an enzyme complex containing cobalt, this element will be
included in the medium at a concentration of a few parts
per million to ensure that it is not scarce. When a medium
contains low concentrations of complex ingredients, it is
important to include a trace element mixture in the fermentation medium.
In fermentations where the ionic strength has to be relatively high, sodium chloride or sodium sulfate is included in
the medium. The insoluble salt calcium carbonate is added
to prevent the fermentation pH from falling below 6.0. As
the pH drops below 6, calcium carbonate dissolves in the
medium, raising its pH. Phosphate salts are rarely used for
buffering in fermentation media because the phosphorus
balance has to be based on the metabolism rather than
on the buffering needs. The soluble calcium salts such as
calcium chloride and calcium acetate are often used to
precipitate out soluble phosphate (in the form of calcium
phosphate) from the media of fermentations in which
product synthesis is strongly inhibited by phosphate. Minerals also serve as precursors in antibiotic fermentations. In
penicillin and cephalosporin fermentations, sufficient sulfate salts have to be included in the medium to supply the
sulfur required for the syntheses of these sulfur-containing
antibiotics. Similarly, chloride salts must be included in the
medium for vancomycin fermentation since the vancomycin molecule contains several chlorine atoms.
46.3.4.
Specialty Chemicals
Several types of specialty chemicals are added to largescale fermentation media. The most important of these
chemicals are the defoamers. The defoamers reduce the
interfacial surface tension between air and water to facilitate bubble coalescence. In the fermentation industry,
silicone and polyol-based defoamers have largely replaced
vegetable oils as defoamers. The advantages of the synthetic defoamers are that they are cost-effective and very
slowly metabolized and do not have appreciable metabolic side effects. The two most popular defoamers in use
in the fermentation industry are polypropylene glycol and
silicone emulsion. The defoamers are generally batched
with the starting medium. In many fermentations, however, it is necessary to supply defoamer throughout the
fermentation cycle to control foam and to control air
holdup. Emulsifiers used in fermentations (such as Tween
and Span) play a role opposite to that of defoamers. They
are added to stabilize small droplets of oily nutrients by
increasing the surface tension between oil and water. The
small droplets have a dramatically increased surface area
and thus allow oily substrates to be more readily utilized
by the fermentation organism. Metal-chelating agents
such as EDTA are often included in fermentation media.
The chelating agents have two diametrically opposed effects. On the one hand, they can tie up metal ions that
are toxic to the microorganism. On the other hand, they
can prevent the precipitation of a required trace metal by
forming a soluble complex. The availability of the metal
to the fermenting microorganism depends upon whether
the microorganism can effectively compete with the complexing agent for the required metal.
An important class of specialty products used in the
fermentation industry is made up of various enzyme preparations. Crude preparations of enzymes such as amylase,
protease, and cellulase are used to precondition the medium. Invariably, these enzymes are used at the mixing
stage before medium sterilization. A partial breakdown
of the starch of medium components such as corn flour
can be achieved by the addition of amylase. The cellulase
complex can be used to reduce the viscosity of a medium
containing a high concentration of ingredients such as soy
or cottonseed flour. Proteases can predigest the medium
proteins before sterilization. Enzymatic pretreatment of a
fermentation medium thus allows a crude and cheaper raw
material to be substituted for a more refined and expensive
raw material. Significant efforts are currently under way to
investigate the pretreatment of cheaper raw materials to
enable their economic fermentation to ethanol or other
biofuels.
46.3.5. Sources of Information on Fermentation
Raw Materials and Microbial Composition
The best source of information on a given class of fermentation raw material is the industry in which it is generated.
Information about such things as the protein, fat, carbohydrate, and mineral contents of various raw materials is
readily available from the supplier of the raw materials.
However, this information is not necessarily generated for
the use of the fermentation industry. It is generated for the
benefit of the primary users, which in most cases are the
animal feed and food industries. As a result, interpretation
of the information for fermentation use is up to the fermentation scientist. For example, while the total nitrogen
value of a grain-based product may be meaningful from the
point of view of a weight gain calculation when the product is fed to a farm animal, it may not necessarily have the
same meaning as the nitrogen available for the fermentation microorganism to grow on. For the same reason, the
carbohydrate value provided by the manufacturer of one
product may be higher than the value provided for a second
product, and yet the second product could have more available carbon for a particular fermentation microorganism.
The information provided by the manufacturer is a good
approximation for the initial evaluation and for preliminary
cost calculations. Actual fermentation experiments are
necessary in all cases to justify a change of raw material. In
recent years, some of the raw materials suppliers have taken
it upon themselves to evaluate their products for various
fermentation processes and publish the results in their own
manuals or in scientific journals.
A list or database of fermentation raw materials is essential for the fermentation medium designer. Such a database
should include medium composition (both molecular and
elemental), but could also include such things as price,
supplier, and availability. Useful lists of such data include
the following.
•
•
Miller and Churchill (12) lists many fermentation raw
materials by their trade names along with their applications in various types of fermentation processes.
Atkinson and Mavituna (1) is a useful source of data
for a range of fermentation situations and has a list of
compositions of fermentation medium components.
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46. raw Materials selection and Medium development
•
•
Solomons (15), an older but classic practical fermentation guide, has a chapter on constituents of fermentation
culture media.
Kennedy and Reader (10) may have general applicability, although this paper describes only raw materials
available in New Zealand.
Many such lists are prepared by the suppliers of fermentation media, such as the Traders’ Guide to Fermentation
Media Formulation (20). A similar information booklet
relating to soy products, titled Soy Protein Products in
Fermentation, is supplied by Cargill, Inc. (3). Knowledge
of suppliers reveals trends in composition and availability
and is particularly useful when it comes to using agricultural by-products for fermentation media.
In the case of some commodity products where there is
intense price pressure on raw-material costs, or when the
goal of the fermentation is to add value to an otherwise
low-value by-product stream, the list of possible medium
components is quite constricted. Usually this means incorporating agricultural products in the fermentation medium
in a very crude state. Data on agricultural products are best
found from the relevant trade organization or from food
nutrition databases. Many countries maintain databases of
food composition, including the United States, Denmark,
Australia, and New Zealand, which profile the main agricultural products of the country concerned. These are
usually conveniently Web searchable; see, for example,
those provided by the U.S. Department of Agriculture
(www.ars.usda.gov/Aboutus/docs.htm?docid=6300) and
Plant and Food Research (www.crop.cri.nz/home/productsservices/nutrition/foodcompdata/fcd-products/fcd-food
-comp-tables.php). Another good source of data on agricultural by-products is the Feed Industry Red Book by Goihl and
McEllhiney (6).
There are also compilations of media that can be useful
for selecting a medium specific for a certain situation. Most
of these compilations are laboratory media, rather than
industrial fermentation media, and thus limited in use for
scale-up. They do, however, provide a useful starting point.
Examples of already specified media sources include Handbook of Culture Media for Food Microbiology (5), Difco & BBL
Manual: Manual of Microbiological Culture Media (2), and
The Oxoid Manual of Culture Media, Ingredients and Other
Laboratory Services (14).
Most practitioners of fermentation medium design have
favorite compositions for specific microorganisms, so soliciting colleagues for a suggested medium for a particular
microorganism is often enlightening.
As discussed earlier in this chapter, the composition
of the microorganism (molecular and elemental) is also
important in medium design for the purpose of calculating
potential biomass and product yield, via mass balance. For
accurate data, it is best to measure the composition for the
microorganism in question.
46.4. GENERAL CONSIDERATIONS
FOR INDUSTRIAL (COMPLEX) MEDIUM
DEVELOPMENT OR IMPROVEMENT
46.4.1. Rationale for Improving a Fermentation
Medium
Designing an improved fermentation medium can be laborious, expensive, open-ended, and time-consuming, involving a large number of experiments. Consequently, it is
important that the effort is justified and, most importantly,
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that the target of the optimization is identified. The main
reasons the fermentation industry designs improved fermentation media are to (i) improve product yield, (ii) decrease
the cost of the medium, (iii) ease product separation, (iv)
avoid the microbe making undesired by-products, (v) reduce waste treatment costs, and (vi) improve robustness
(the ability of a medium to repeatedly perform well at large
scale). It is very important at the start of a medium design
campaign to clearly identify which of these reasons is the
primary target of the effort.
In a typical industrial antibiotic fermentation, improving product yield always has a larger impact on the overall
process cost than does simple medium cost reduction. Very
often, yield improvement not only improves the economy
of the fermentation process itself but also has beneficial effects on downstream processing. The product-to-impurity
ratio increases as the fermentation yield increases, making
the recovery process more efficient. In some mature fermentations, productivity improvement beyond a certain
level is difficult to attain due to genetic limitations or
the inability of the microorganism to tolerate increasingly higher concentration of the product. In such cases,
fermentation raw-material cost reduction alone can be the
major component of the overall cost reduction efforts. The
value of the final product and the volume of the product
produced are other important considerations. First, consider the final product value relative to the cost of the raw
materials used. In the fermentation industry, the contribution of fermentation raw materials to the overall production cost may vary from as little as 5% (for example, the
production of high-value biological agents such as interferon or the production of steroids) to as much as 50% (for
example, the production of commodities such as ethanol).
The scientist working on the former type of product has
much greater flexibility in selecting raw materials, since
the overall production cost is not appreciably increased
by introduction of a relatively costly raw material. The
goal here is to reduce the overall cost by increasing the
fermentation yield. In the latter case, however, the incremental cost increase due to the introduction of a new raw
material has to be more than compensated by the increase
in yield and product quality. The agricultural commodity
products and by-products from the brewery and corn wetmilling industries are the typical raw materials used in
fermentation processes for low- and medium-value products such as organic acids and well-established antibiotics.
On the other hand, exotic raw materials such as refined
yeast extracts and exotic growth factors can be cost-effective in fermentation processes of high-value products
such as biological peptides. The usage rate of a given raw
material and the overall volume of the fermentation broth
processed also have to be taken into account for medium
development decisions. If an ingredient is used at a few
parts per million, its unit cost does not significantly affect
the overall process cost. If the volume of the fermentation
broth is very large, however, the overall cost may still be
significantly affected.
The availability of a given raw material in a given geographic location is another consideration. Should a specific
material be shipped long distance, or should the medium
formulation be changed so that a readily available material
can be used in its place? This depends largely upon how sensitive the fermentation yield is to the type of material used.
While a readily available raw material may give a somewhat
reduced yield, in the long run it may be more cost-effective to standardize the medium with that material than to
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biological engineering and scale-up of industrial processes
depend upon a material that gives higher yield but may be
subject to supply disruption. On the large scale, supply disruption is a crucial issue. Access to at least two suppliers of
any particular component in the medium is recommended.
This recommendation prevents a large disruption should
one supplier suddenly no longer supply a given component or go out of business. In the case of some agricultural
products, adverse weather can disturb supply. Agricultural
products are also susceptible to price fluctuations. A rapid
rise in cost of one component may mean its removal from
the fermentation and substitution with another. One recent
example of such price fluctuations was caused by the rapid
rise in the use of biofuels, disturbing supply and prices of
agricultural commodities. It is a useful exercise to rank each
substrate on a price/kilogram of carbon and price/kilogram
of nitrogen basis for comparison. In this way critical prices,
where an alternative substrate becomes more cost-effective,
can be identified and market prices tracked. A new component validation procedure ensures that new components
can quickly be given an end-use test to confirm their performance and robustness.
Other factors to consider are whether the quality of the
material will be adversely affected during long-distance
shipping and/or prolonged storage. Raw materials such as
yeast paste and corn steep liquor are not stable enough for
prolonged storage. On the other hand, raw materials with
low moisture content such as cottonseed meal, soy flour,
and spray-dried yeast are reasonably stable over long periods
of storage.
46.4.2.
Nature of Fermentation Raw Materials
Most raw materials used in the fermentation industries
are not designed for that use. They are generally designed
to supply commodities for the food and feed industry.
Thus, soy meals, cottonseed meals, and corn gluten meals
are designed primarily as animal feed protein sources.
Various yeast products are designed for both human food
and animal feed applications. Corn syrups of different
levels of hydrolysis are made for application in the foodprocessing industry. Since the fermentation industry is
not the primary user of these raw materials, the industry
does not have much control over their processing and
the resulting quality from the point of view of their use
in fermentation processes. Also, agricultural products are
subject to variation due to growing seasons, soil conditions, and storage conditions. In short, raw-material variability is the rule rather than the exception. In medium
design, then, it is necessary to use multiple sources of the
same class of nutrient to reduce process variability. Thus,
including two complex nitrogen sources in the medium
formulation is more desirable than depending upon a
single ingredient. It is also recommended that several lots
of the same raw material be tested before settling on a
given medium formulation. If the product yield varies excessively due to lot-to-lot variability, it is better to avoid
that raw material in the medium formulation altogether.
Crude complex raw materials are more likely to exhibit
greater variability in composition compared to synthetic
medium components.
At this point, it should be noted that water used to
prepare fermentation medium is the major component
of the medium. In large fermentation plants, this water
is usually not distilled or deionized water, as may be the
case in the laboratory. As a result, certain metal ions and
organic components that come dissolved in the water as
impurities become part of the fermentation medium. These
impurities and their concentrations may vary on a seasonal
basis. In addition, the profile of inorganic and organic
components that come with the water may vary when the
municipal water treatment plant experiences upsets in its
operations. Many fermentation plants use readily available
water from adjacent water sources such as lakes, rivers, or
deep wells with minimal pretreatment. These water sources
are also subject to seasonal variability. Water quality is
an important variable when fermentation processes are
scaled up from the laboratory, where deionized or distilled
water may be used. The water quality is also an important
consideration when fermentations involving identical raw
materials perform differently at differing physical plant locations. Most fermentation plants monitor the water quality only superficially, and it is seldom known which water
quality parameters are important for a given fermentation
process.
46.5. GENERAL GUIDELINES FOR
FERMENTATION MEDIUM DEVELOPMENT
46.5.1. Seed Medium and Product Synthesis
Medium
Generally, the purpose of the seed culture is to grow cells
as fast as possible on the basis of predefined criteria such as
dissolved-oxygen level, oxygen uptake rate, or centrifuged
cell volume. This can be readily achieved by supplying
the required nutrient for growth without regard to the
product formation needs. When developing a medium for
the production stage of a process, the selection of medium
components and optimization of their concentrations in
the medium are more involved. The objective is not only
to develop cell mass but also to synthesize the product
at the highest rate possible. The cell density attained,
the growth rate during the cell growth, the fermentation
time, and the subsequent maintenance metabolism are all
important factors in maximizing product formation. The
rate of cell growth often can be controlled by controlling
the level of readily available nutrients such as glucose,
amino acids, and soluble phosphate and by controlling the
growth temperature. The slow-growth and maintenance
metabolism during the product synthesis phase of the
fermentation process is generally controlled by supplying
additional nutrients slowly. This controlled nutrient feed
is usually composed of glucose or vegetable oil. In some
cases, ammonia or complex nitrogen sources are also supplied during this phase. Another way of controlling the
slow-growth and maintenance phase of the fermentation
process is to include in the medium a carbon (or nitrogen)
source that is only gradually utilized by the organism. Often, carbohydrates such as lactose or starch are used for this
purpose. The organism being cultivated must produce specific enzymes such as b-galactosidase or amylase to be able
to utilize these carbon sources. Various oils are frequently
added as the source of carbon that is gradually consumed
by microorganisms exhibiting lipase activity. Coarse raw
materials such as soybean grits and corn gluten meal are
used to supply slow-releasing nitrogen. Enzymes such as
cellulase and protease must be induced for the organism to
utilize these coarse nitrogen sources. In addition to maintenance nutrients, some secondary-metabolite fermentations
require the addition of precursor compounds. For example,
the precursors phenylacetic acid and uracil are added to
fermentations of the antibiotics penicillin and nikkomycin, respectively.
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46. raw Materials selection and Medium development
46.5.2. Using Laboratory Fermentation Medium
as the Starting Point
Industrial fermentation organisms are generally highly mutated organisms that are developed in strain development
laboratories over many years. The fermentation conditions
under which these organisms have been selected must be
taken into consideration during medium development
work. If a strain has been selected with a laboratory fermentation medium that is based on cottonseed meal as
the primary nitrogen source, it may not perform well in a
medium based on corn steep liquor as the primary nitrogen
source. This is not to say that more closely related medium
ingredients such as soy flour and peanut meal may not give
yield improvements. The relationship between the laboratory carbon source and the carbon source used in the largescale fermentation is often not straightforward. Because
in shake flask fermentations external pH control is not
possible, a readily utilized carbon source such as glucose
is very seldom used unless the medium is heavily buffered.
Typically, a carbon source such as sucrose, lactose, dextrin,
or starch is used to maintain the pH in a reasonable range.
When these processes are scaled up, similar complex carbohydrates are initially used in a batch mode. As the process
is developed further, however, they are often replaced
with an external feed of a readily utilizable carbon source
such as glucose, accompanied by pH control. A similar
situation can also arise with regard to simple nitrogen
sources. Although nitrates or amino acids have to be used
in a laboratory fermentation medium for the purpose of pH
balancing and slow nitrogen release, they can be replaced
in large-scale fermentations with more readily available
and cheaper materials such as ammonium sulfate or ammonia, with appropriate control mechanisms. The overall
efficiency of nutrient utilization may also change when
the fermentation process is scaled up to large fermentors,
in which the agitation and aeration conditions are more
intense than those in shake flasks. More often than not,
the nutrient requirement increases when going from shake
flasks to large fermentors.
46.5.3. Considerations of the Fermentation
Medium as a Whole
A fermentation medium is typically prepared by dissolving
or suspending various raw materials in water. Before the
medium is inoculated with the desired microorganism, it
is heat sterilized. The batch sterilization involves heating
the medium to over 121°C for a period ranging from 30 to
60 min. Continuous sterilization is carried out by rapidly
bringing up the temperature to 145 to 155°C and holding
it at that temperature for 5 to 10 min. This heat sterilization of a mixture of ingredients in water has a profound
effect on the resulting fermentation medium. A number
of chemical and physical changes occur during sterilization. Insoluble ingredients such as grain flours and meals
are partially solubilized. Macromolecules such as proteins
and starch are partly degraded to more soluble and readily
metabolizable lower-molecular-weight peptides and oligosaccharides. The inorganic components of the medium
react among themselves and with organic components
to give new compounds. For example, various metal ions
complex with protein molecules to alter protein solubility,
organic phosphorus compounds release phosphate into the
medium, dissolved phosphorus is precipitated as insoluble
metal phosphates, etc. In some cases, the heat sterilization
generates toxic chemicals from relatively benign medium
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ingredients. A well-known example of this toxicity is the
Maillard reaction between reducing sugars and amino compounds to give growth-inhibiting amino sugars. To prevent
this reaction, reducing sugars such as glucose are sterilized
separately from the medium containing amino acids and
ammonia. The two components of the medium are mixed
after they are cooled to about 40°C. Since various medium
components interact during sterilization, it is important to
examine the effect of an ingredient being added or removed
on the overall chemistry of the medium. The organism
may not require calcium salt for growth or for product
formation. However, calcium may play a critical role by
precipitating out excess phosphate from the medium in the
form of insoluble calcium phosphate and allowing a phosphate-regulated product to be synthesized. The elimination
of soluble phosphate will also change the medium’s buffering capacity. The pH of the medium during sterilization is
important because the chemical reactions occurring in an
aqueous medium are affected by pH. The pH can have an
effect on both the rates of reactions and the equilibrium
composition. For this reason, it is generally necessary to experiment with sterilization pH to optimize the performance
of the medium under development. It is well known that by
manipulating sterilization pH, one can increase or decrease
protein solubilization from a medium containing insoluble
protein sources such as grain flours and meals.
Sterilization heat damage to a medium can have a
significant impact on fermentation medium performance.
Unfortunately, heat input to a fermentation medium during
sterilization can vary considerably on scale-up, depending
on fermentor geometry. One way to quantitatively track the
heat input as scale is varied is the use of the del factor (or
Ro) calculation (18). Del factor can be used to track variations in heat input between sterilization batches, should a
medium be unavoidably sensitive to such damage.
Some components of the medium may have an indirect
effect even in the absence of heat sterilization. For example,
seemingly inert oils and defoamers may create micelles in the
broth that solubilize proteinaceous components and fats that
may otherwise be unavailable for metabolism. Some surfaceactive agents have no metabolic effect but may have substantial effects on the oxygen transfer characteristics of the
fermentation broth by changing the surface tension at the
air-liquid interface. Many fermentation media containing
complex proteins tend to foam heavily during sterilization,
and addition of defoamer may be necessary even though the
fermentation process itself does not require foam control
chemicals. On rare occasions, the order of addition of various ingredients when the medium is prepared, the temperature at which the presterilized medium is prepared, and the
length of time the medium is held before sterilization will
affect the performance of the fermentation process.
Particle size can have a significant effect on fermentation
medium performance. Large particles have a smaller surface
area per unit volume than small particles, and this means
that they may dissolve or get degraded at a slower rate during
the fermentation, affecting fermentation kinetics. Calcium
carbonate is particularly susceptible to this effect. Large
particles also require a longer time to sterilize, meaning more
heat damage to other components in the medium. Solid
substrates also interfere with the common DCW cell mass
assay, and one of the advantages of a synthetic medium is the
ability to conduct meaningful DCW cell mass assays.
Regulatory preferences also play a part in fermentation medium design. Some companies may wish to avoid
using all animal-derived products due to the potential
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biological engineering and scale-up of industrial processes
contamination by self-replicating proteins referred to as
prions. Strong evidence supports prions’ being responsible
for causing bovine spongiform encephalopathy, a transmissible spongiform encephalopathy, in cattle. Other companies may accept the use of milk-based products, e.g., casein
hydrolysate, or fish-derived products, e.g., fish meal.
When designing a fermentation medium, it is worthwhile doing an elemental mass balance, not only to see
that sufficient components are present to achieve a desired
yield but also to inspect the medium for overdosing. Too
high a concentration of some minerals may be toxic. Copper is an example of a mineral of which it is easy to add too
much. The exact amount that a microbe will tolerate can
be strain specific. Corrosion of equipment may inadvertently add minerals to toxic levels. For this reason, copper
pipes contacting the medium are to be avoided. Corrosion
may also present an issue in the special case of growing
marine microbes. Some marine microbes need high levels
of sodium chloride in the fermentation medium, which can
provide a significant corrosion challenge for stainless steel
equipment.
Often forgotten in fermentation medium design is the
fact that oxygen is a metabolic requirement and that carbon
dioxide can dissolve in the medium and become inhibitory. While developing a medium at laboratory level, poor
results may be due to poor oxygen transfer rather than the
components in the medium. Another trap when developing
a medium at small scale is the appearance of wall growth.
This will invalidate results, as product formation (or lack
thereof) in this wall growth is not representative of what
will happen in a large-scale fermentor (where wall growth,
if present at all, will be only a small fraction of the total
fermentation volume). Wall growth on the small scale can
be greatly reduced by adding a small amount of a growth
dispersion agent for filamentous microorganisms, such as
Junlon polyacrylic acid (7).
Because the fermentation medium after sterilization
(and hence after the chemical and physical changes have
taken place) is the real medium in which the organism of
interest is to be grown, it is important to characterize the
sterilized medium. Certain overall indices such as soluble
nitrogen, reducing-sugar equivalent, and soluble phosphate
are often used to characterize the sterilized fermentation
medium. However, these indices give only a gross measure
of the properties of the medium. Only by understanding the
chemical and physical phenomena taking place in the medium during batching, sterilization, and the fermentation
process itself can one truly master the art of fermentation
medium development.
Last, it is important to ask, when are medium design
efforts sufficient? Usually some form of medium design
continues during the life of the product due to the introduction of new strains from a strain improvement program, the
necessity of further yield improvements, or the replacement
of components for some reason. The time to stop is when
resources run out or when no changes seem to improve the
best medium. It is important not to overinvest in medium
design, and one tool useful in assessing this is to plot the
number of media tested versus the maximum performance
to date. Typically such a curve follows asymptotic behavior
quite quickly (especially if statistical experimental design is
used), which indicates visually when the point of diminishing returns is reached (11).
Fermentation medium design is both an art form and a
logical science, and this is what makes it a challenge.
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