Industrial Fermentation Processes

T M Anderson, Archer Daniels Midland BioProducts Plant, Decatur, IL, USA

2009 Elsevier Inc. All rights reserved.

Defining Statement
Introduction
History
Culture Selection and Development

Glossary
aseptic The absence of contaminating
microorganisms.
auxotrophy The inability to synthesize a substance
necessary for growth.
bacteriophage Viruses that infect and replicate within
bacteria. Often referred to as phage.
bioreactor A closed vessel used for fermentation or
enzyme reaction.
BOD (biological oxygen demand) The amount of
oxygen required by microorganisms to carry out
oxidative metabolism in water containing organic
matter, such as sewage.
chemostat A continuous culture system that maintains
microorganisms in exponential growth phase for an
extended time, usually by limiting an essential nutrient.
constitutive Expression of a gene without regulation of
RNA polymerase. Some production cultures are
mutated to constitutively express genes critical for
making the desired product.
corn steep liquor A by-product of the corn wet milling
process, rich in organic nitrogen and other nutrients;
used as an inexpensive nutrient source in fermentation.
dissolved oxygen The quantity of oxygen dissolved in
fermentation broth, expressed as percent of saturation.

Abbreviations
BOD
DO
GMO
MSG

biological oxygen demand

dissolved oxygen
genetically modified organism
monosodium glutamate

Defining Statement
Fermentation has been used since before recorded history
to make food products, and today hundreds of products are

Process Development and Scale-Up

Large-Scale Operation
Future Prospects
Further Reading

feedback inhibition The inhibition of an enzyme in a

metabolic pathway by the end product of the pathway.
GMO (genetically modified organism) Any organism
that has been modified using genetic engineering
methods.
induction Expression of a gene for an enzyme
stimulated by another compound, often a substrate or
structurally similar compound.
inoculum The culture used to start a fermentation.
mass transfer Molecular and convective transport of
atoms and molecules within physical systems, such as
the transfer of oxygen from air bubbles to the
fermentation broth.
seed fermentor A growth stage fermentor, used to
inoculate a production fermentor.
sterilization The process of destroying all living
organisms in or on a material.
TCA cycle (tricarboxylic acid cycle or Krebs
cycle) The cycle by which carbohydrates are
completely oxidized to carbon dioxide and water.
thermophile A microorganism that grows best at
temperatures greater than 50  C.
ultrafilter A filter with very small pore size, usually
smaller than a molecular weight of 100 000 Da.

NRRL
OUR
PLA
TCA cycle

Northern Regional Research Laboratory

oxygen uptake rate
polylactic acid
tricarboxylic acid cycle or Krebs cycle

made by industrial fermentation. Fermentation development involves selection and genetic development of the
production culture, process development, and scale-up.
Once in production, physical and chemical parameters

349

350 Applied Microbiology: Industrial | Industrial Fermentation Processes

must be tightly controlled for optimum productivity, and

contamination control is vital to successful fermentation
operation.

processes are generally much slower than

Microbial
chemical processes, increasing the fixed costs.
processes are subject to contamination by
Microbial
competing microbes, requiring the sterilization of raw

Introduction
Many products are made by industrial fermentation
today, including amino acids, enzymes, organic acids,
vitamins, antibiotics, solvents, and fuels (Table 1). Most
industrial fermentations use bacteria, yeast, and fungi, but
some processes use algae, plant, or animal cells.
There are several reasons that certain products are
made by fermentation:

Complex molecules that occur naturally antibiotics,

enzymes, and vitamins are too complex to produce
chemically.
Optically active compounds, such as amino acids and
organic acids, are difficult and costly to make chemically.
Food products alcoholic beverages, vinegar, flavors,
and fragrances that could be more economically
produced chemically are produced by fermentation
so that they can be labeled as natural products.
Fermentation primarily uses renewable resources
instead of petrochemicals.
Reaction conditions are mild and in aqueous media.
Most reaction steps occur in one vessel.
The by-products of fermentation are usually environmentally benign compared to the organic chemicals
and reaction by-products of chemical manufacturing.
Often, the cell mass and other major by-products are
highly nutritious and can be used in animal feeds.

There are some disadvantages to making products by

fermentation:
products are made in complex solutions at low
The
concentrations compared to chemically produced compounds; thus purification is often difficult and costly.

materials and the containment of the process to avoid

contamination.
Most microorganisms do not tolerate wide variations in
temperature and pH and are also sensitive to upsets in
the oxygen and nutrient levels. Such upsets not only
slow the process, but are often fatal to microorganisms.
Thus nutrients, pH, oxygen, temperature, and agitation require careful monitoring and control.
Although usually nontoxic, waste products are high in
biological oxygen demand (BOD), requiring extensive
sewage treatment.

Fermentation products are derived from a variety of

cellular functions. Some products, such as primary
metabolites and whole cell products, are produced during the growth phase. Other products, such as secondary
metabolites, energy storage compounds, and polymers,
are generally produced after the growth of the culture.
Some other products depend on their metabolic function or, for genetically engineered proteins, on whether
the production is induced or constitutive. Examples of
cellular activities and their related products are as
follows:
metabolites ethanol, lactic acid, acetic acid,
Primary
amino acids, and citric acid
cell products bakers yeast and brewers yeast,
Whole
and starter cultures
metabolites antibiotics, antitumor com Secondary
pounds, antiviral compounds, and bioinsecticides
storage compounds glycerol, polymers, oils,
Energy
and polysaccharides
extracellular and intracellular enzymes, sin Proteins
gle cell protein, and genetically engineered proteins.

Table 1 Fermentation products

Category

Examples

Functions

Organic
acids
Organic
chemicals
Amino acids

Lactic, acetic, formic, citric, and acrylic

acids
Ethanol, glycerol, acetone, butanediol,
propylene glycol
MSG, lysine, threonine, tryprophan,
phenylalanine
Penicillin, streptomycin, tetracycline,
chloramphenicol
Amylases, cellulases, glucose isomerase,
proteases, lipases
Xanthan gum, dextran, poly--hydroxybutyrate,
poly-3-hydroxyalkanoates
Vitamin B12, biotin, riboflavin
Yeast, lactic acid bacteria

Food acidulants, textiles, tanning, cleaners, detergents,

plastics, organic chemicals
Fuels, solvents, plastics, rubber, chemicals,
antifreeze, cosmetics
Animal feeds, flavors, sweeteners, dietary supplements

Antibiotics
Enzymes
Biopolymers
Vitamins
Cell mass

Human and animal health care

Grain processing, detergents, cheesemaking, tanning,
juice processing, high fructose corn syrup
Stabilizers and thickeners in foods, oil extraction,
plastics
Nutritional supplements, animal feed additives
Single cell protein, bakers and brewers yeast,
starter cultures

Applied Microbiology: Industrial | Industrial Fermentation Processes

History
Humans have used fermentation from before the beginning of recorded history to make products for everyday
use. For millennia, the primary use of microbial processes
was to preserve or alter food products for human consumption. Fermentation of grains and fruit produced
bread, beer, and wine, which retained much of the nutrition of the raw materials while keeping the products from
spoiling. The natural yeasts that caused the fermentation
added some vitamins and other nutrients to the bread or
beverage. Lactic acid bacteria fermented milk to yogurt
and cheeses, extending the shelf life of milk products.
Other food products were preserved or enhanced in flavor
by fermentation, such as pickled vegetables and the fermentation of tea leaves and coffee beans.
Fermentation was more art than science until the second
half of the nineteenth century. A batch was begun with
either a starter culture a small portion left from the
previous batch or the cultures naturally residing in the
product or vessel. The notion that microbes were responsible for fermentations was not introduced until 1857, when
Louis Pasteur published a paper describing the cause of
failed industrial alcohol fermentations. He also quantitatively described microbial growth and metabolism for the
first time and suggested heat treatment (Pasteurization) to
improve the storage quality of wines. This was the first step
toward sterilization of a fermentation medium to control
contamination. The use of pure yeast cultures for beer
production was introduced in Denmark in 1883 by Emil
Christian Hansen. Beer and wine were produced on a
relatively large scale at the beginning of the eighteenth
century to satisfy the demands of growing urban populations. In the mid-nineteenth century, the introduction of
denaturation freed ethanol from the heavy beverage tax
burden so that it could be used as an industrial solvent
and fuel.
The first large-scale aseptic fermentation was acetone
butanol fermentation, which both Great Britain and
Germany pursued in the years preceding World War I.
The initial objective was to produce butanol as a precursor
for butanediol for making synthetic rubber. After the war
began, Great Britain was cut off from its supply of acetone
for munitions manufacture, which had previously been
imported from Germany, shifting the focus to acetone.
As the process was developed and scaled up, it was
found that the producing culture, Clostridium acetobutylicum,
would become overwhelmed by the competing bacteria
introduced from the raw materials. The culture medium
had to be sterilized and the process run under aseptic
conditions. All penetrations on the reaction vessel were
steam sealed to prevent contamination. Production eventually took place in Canada and the United States due to
the availability of inexpensive raw materials.

351

In 1919, the 18th amendment to the constitution prohibited alcoholic beverages in the United States, making
facilities available for other uses. The emphasis in fermentation shifted to organic acids, primarily lactic acid, and citric
acid, which could be produced in the nonsterile beer and
wine fermentors. As part of an effort to find uses for agricultural products during the depression, the USDA
Northern Regional Research Laboratory (NRRL) pioneered the use of surplus corn products, such as corn steep
liquor, and the use of submerged fungal cultures in fermentation. Previously, fungi had been grown on solid media or
on the surface of liquid media. This set the stage for largescale production of penicillin, discovered in 1929 in Britain,
developed in the 1930s and commercialized in 1942 in the
United States. It was the first so-called miracle drug, routinely curing bacterial infections that had previously caused
serious illness or death. The demand was very high during
World War II and the following years. Penicillin was initially produced as a surface culture in quart milk bottles, but
the cost, availability, and handling of bottles severely limited
the expansion of production. Scientists at the NRRL, with a
new production culture discovered on a moldy cantaloupe,
developed submerged culture fermentation. This led to
major increases in productivity per unit volume and the
ability to greatly increase the scale of production by using
stirred tank bioreactors. The success of penicillin inspired
pharmaceutical companies to launch massive efforts to discover and develop many other antibiotics in the 1940s and
1950s. Most of these fermentations were highly aerobic,
requiring high aeration and agitation. As the scale of production increased, mass transfer became limiting. The field of
biochemical engineering emerged as a distinct field at this
time to study mass transfer problems in fermentation and to
design large-scale fermentors capable of high transfer rates.
In the 1960s, amino acid fermentations were developed
in Japan. Initially, monosodium glutamate (MSG) was
produced as a flavor enhancer to replace MSG extracted
from natural sources. Other amino acid fermentations
were developed using cultures derived from glutamic
acid bacteria. Commercial production of enzymes for
use in industrial processes began on a large scale in the
1970s as well, accounting for 80% of all enzymes in
commercial use. The discovery of the tools of genetic
engineering in the 1970s expanded the possibilities of
products made by fermentation. In 1977, insulin was the
first genetically engineered product to be commercialized. Since then, many genetically engineered products
have been produced on a large scale.

Culture Selection and Development

Any new fermentation process begins with the selection of
the culture to be used for production, which begins once the
desired product or microbial activity is defined. It is rare that

352 Applied Microbiology: Industrial | Industrial Fermentation Processes

cultures isolated from the environment or obtained from a

culture collection produces the product cost effectively,
often making only very small quantities. Increasing the
productivity of the initial isolates requires a program of
genetic development.

Selection
Candidate cultures should produce, or have the metabolic
potential to produce, the desired product. Other attributes
are important, such as substrate specificity, nutritional
requirements, physiological characteristics (pH and temperature ranges, oxygen requirements, shear sensitivity,
etc.), fermentation by-products, effects on downstream
processing, and environmental and health effects.
Literature searches can narrow the range of cultures to
be screened, saving valuable time in culture selection.
Culture collections often have cultures with some of the
desired characteristics. However, it is sometimes necessary to isolate cultures from the environment.
Determining where organisms with the desired characteristics live and how to separate them from the other
cultures present takes considerable care. A thorough
understanding of the physiology of the desired microorganism is necessary to design an isolation and selection
strategy. Successful isolation of useful cultures requires a
combination of several enrichment and selective methods.
A system of storing and cataloging potentially useful
isolates is crucial, so that commercially viable cultures
are not lost. After initial screening, there are usually many
potential isolates. Secondary screening eliminates false
positives and evaluates the potential of the remaining
candidates. The list of candidates is narrowed as much
as possible using mass screening methods, such as agar
plates with selective growth inhibitors or metabolic indicators. Advances in robotics and spectrophotometric
analysis have allowed the screening of thousands of
potential cultures in multiwell plates. Once potential
candidates have been reduced to a more manageable
number, more detailed studies are performed in shake
flask culture, which is time-consuming and expensive
compared to mass screening. Finally, the initial isolates
are reduced to a few promising candidates.

Development
There is a vast array of tools available to geneticists for
improving the production capabilities of cultures. These
include classical genetics (pre-genetic engineering), gene
cloning, directed mutagenesis, directed evolution, and
metabolic flux analysis. The goal of genetic modification
is to eliminate or reduce unwanted metabolic pathways,
enhance desired pathways, modify substrate or product
specificity, or reduce product feedback inhibition.

Classical mutation and selection methods have been

used for many decades and are still in use, especially if
little is known about a cultures genetics. Classical genetic
methods are also used when the use of genetically modified organisms (GMOs) are restricted. The culture is
subjected to random mutagenesis by ultraviolet light,
ionizing radiation, or chemical mutagens and then either
grown in the presence of a selective growth agent, such as
a chemical analogue to the final product, or grown in the
presence of a growth or production indicator. The promising survivors are isolated and tested. This is usually an
iterative process; mutant strains are screened, mutagenized, and selected several times, often using stronger
selective agents, until a culture with commercial potential
is obtained. Selecting for improved growth rate, substrate
utilization, or tolerance to inhibitors such as high product concentrations or high osmotic pressure can be
accomplished without mutagenesis by repeated transfer
of a culture in shake flasks under the selective pressure
or by growing the culture in chemostat culture with
either increasing dilution rate or increasing inhibitor
concentration.
Since the 1970s, genetic engineering techniques have
been used for strain improvement. The methods have
become increasingly sophisticated, from simple cutand-paste gene cloning to protein engineering and directed evolution. Inserting genes into plasmids improved the
ease with which genes from one culture are transferred to
another and allowed the production of human and other
mammalian proteins such as insulin and interferon in
microbial fermentations. Genes inserted into plasmids
with high copy numbers or coupled with strong transcriptional promoters dramatically improved production of
many proteins and enzymes.
Production cultures developed from classical mutagenesis often have hundreds of silent mutations for
every desired mutation. Although these mutations have
little individual effect, they cumulatively reduce the
growth and robustness of the strain. Protein engineering
is the site-specific mutagenesis of enzymes based on
detailed knowledge of enzyme structure and sophisticated
targeted mutagenesis techniques. This increases the efficiency of mutagenesis programs and eliminates the
deleterious effects of the random or shotgun mutagenesis
used in classical genetics. If little is known of the enzyme
structure, directed enzyme evolution can be used to
improve or change enzyme function by randomly mutagenizing the gene, creating a library of mutants. The
mutants are inserted into a host culture and the resulting
colonies are screened for the properties of interest. This
process is repeated multiple times to obtain the desired
characteristics. Mutants with desirable traits can also
be recombined to further alter enzyme function. Both
enzyme engineering and enzyme evolution can be used
in conjunction with metabolic flux analysis, which uses

Applied Microbiology: Industrial | Industrial Fermentation Processes

knowledge of the reactions within a cell to calculate the

flow of carbon and energy within the system. This can be
used to identify targets for genetic engineering. After a
process is successfully brought to production, culture
improvement is ongoing to improve profitability and
maintain a competitive advantage.

Process Development and Scale-Up

Process development usually overlaps culture development. This compresses the timeline from initial concept
to production. In addition, it is often found during process
development that additional genetic improvements of the
culture are required. The purpose of process development and scale-up is to formulate media, optimize
culture conditions, and determine the biochemical parameters used to design full-scale bioreactors.
Fermentation Development
The early stages of process development are usually
performed in shake flasks, where the nutritional requirements of the culture are determined and potential media
components are screened. Initial growth and production
studies can be performed in shake flasks as well, but there
are limitations to shake flask culture. Parameters such as
pH, oxygen content, and other environmental factors
cannot be easily monitored and controlled and mass
transfer studies are difficult.
The next stage of process development is performed in
laboratory-scale fermentors. Here, the media components
and concentrations are optimized, nutrient feed rates and
parameters such as optimum pH, oxygen uptake, heat
generation, growth, and production rates, as well as sensitivity to nutrient and by-product concentrations, broth
viscosity, and shear forces, are determined. This information is used to design the bioreactor and calculate the
costs and potential yield of the process.

aerobic conversion of carbohydrate to biomass and product is:

CHa Ob carbohydrate
AO2 B NH3

! Yx=s CH O N biomass

Yp=s CH1 O1 N1 product Yco2 =s CO2
DH2 O

The terms Yx/s, Yp/s, and Yco2/s are the yield coefficients
for cell mass, product, and CO2, respectively. In most
industrial fermentations, the carbon usually comes from
carbohydrates such as sugars or starches. The nitrogen is
from a variety of organic and inorganic sources and from
the ammonia added to control the pH. Phosphate, sulfur,
and magnesium are added as salts or in complex nutrients.
The micronutrients are often derived from the water or
other raw materials, or added as mineral salts. Some commonly used fermentation substrates are shown in Table 2.
Many microbes are auxotrophs for specific growth
factors, such as vitamins or amino acids, and will be
growth limited without adequate quantities of the compound in the medium. This is usually evident by the
cessation of growth and production in the presence of
adequate substrate and oxygen. Other cultures may not
have an absolute requirement for a growth factor, but
grow at a reduced rate in the absence of the specific factor.
In many cases, the exact requirements are not known, but
complex substrates are required for optimal growth. It
is often necessary to screen a variety of potential
nutrients and combinations of nutrients to satisfy the
requirements and minimize raw material costs. Another

Table 2 Fermentation raw materials

Function

Raw material

Carbon

Glucose
Sucrose
Lactose
Corn syrup
Starch
Ethanol
Paraffin
Vegetable oils
Beet molasses, cane
molasses
Ammonia
Urea
Ammonium nitrate
Ammonium sulfate
Soy flour
Corn or wheat gluten
Cottonseed oil
Casein
Brewers or bakers yeast
Yeast extract
Corn steep liquor
Distillers dry solubles

Media Development and Optimization

The development of a suitable economical medium is a
balance between the nutritional requirements of the
microorganism and the cost and availability of the medium
components. The chemical constituents of the medium are
determined by the composition of the cell mass and product, the stoichiometry of cell and product formation, and
yield coefficients, which can be established from shake
flask or laboratory-scale fermentor experiments. On a
dry weight basis, 9095% of microbial biomass consists
of carbon, oxygen, hydrogen, nitrogen, sulfur, magnesium,
and potassium. The remaining 510% is microelements
(required in small amounts), primarily calcium, manganese, iron, copper, and zinc. The general equation of

353

Carbon, vitamins, micronutrients

Inorganic nitrogen

Inorganic nitrogen, sulfur

Organic nitrogen

Organic nitrogen, vitamins,

micronutrients

354 Applied Microbiology: Industrial | Industrial Fermentation Processes

The purpose of the scale-up phase is to further refine the

fermentation parameters, bioreactor design, and economic calculations as well as to determine whether
there are any unexpected consequences of increasing
scale. A variety of methods for scale-up of bioreactors
have been applied over the years, generally attempting
to hold critical biochemical engineering factors, such as
oxygen transfer rate, agitation power per volume, impeller tip speed, or equal mixing times, constant. Each has
limitations in predicting the effect of scale-up on the
process. It is more difficult to predict scale-up of a biological process than of a chemical process from laboratory
and pilot plant studies, due to the complexity of reactions
and interactions that occur in the bioreactor. There are
often unforeseen consequences of changing the scale of
operation due to the effect of heat and mass transfer on
microorganisms. For example, mixing times in a laboratory or pilot scale is a few seconds, while in large-scale
fermentors mixing times can be several minutes. The
average conditions are the same in both fermentors, but
an individual cell encounters a variety of suboptimal
conditions for a significant period of time in the largescale fermentor. Also, as the fermentor size increases, the
heat generation increases proportional to the volume
while cooling capacity increases proportional to surface
area, requiring internal cooling coils in larger vessels to
supplement the water jacket. This can further aggravate
mixing problems. The properties of the fermentation
broth (viscosity, osmotic pressure, concentrations of substrate, product, and waste product) and gas/liquid
interactions (gas lineal velocity, surface tension, and pressure gradients) are also scale dependent. Thus,
fermentation scale-up often becomes highly empirical
and based on the experiences and training of the scientists
and engineers involved. The cost and risk of scale-up are
higher for biological systems than for other chemical
systems due to the intermediate scale pilot plant steps
required to successfully predict the outcome of full-scale
operations. Some of this cost can be reduced by using
existing or rented facilities to test the process, or by using
seed fermentors (typically 510% as large as production
fermentors) for the final pilot scale.
An unstable culture can cause serious disruption to
production in a large-scale fermentation plant due to

(a)
1.E+00

Fraction of parent organism

Scale-Up

the increase in the number of generations required for

full-scale operation. Most commercially used microorganisms are mutated and selected for increased product
yield and rates of production, which decreases the growth
rate and hardiness of the culture. Therefore, a variant that
either reverts to a previous condition or short-circuits the
selected pathways by additional mutations will have a
competitive advantage over the production strain. The
percent of variants in the population in a given generation
depends on both the rate at which variants appear and the
relative growth rate of the variant to the parent population as shown in Figure 1. In addition, variant strains are
usually less sensitive to adverse conditions (deviations in
temperature or pH, nutrient quality, or anaerobic conditions) than parent strains. A production strain that appears
stable for the required number of generations under
laboratory conditions may exhibit instability when introduced to a full-scale plant. It is important to test the
stability of potential production strains under conditions
that are as similar as possible to conditions expected in the
production environment. It is also important to establish a
system of culture storage and management that minimizes
the number of generations required to reach full-scale

= 0.001

1.E01

= 1.5
=2
=4

1.E02

1.E03

1.E04

(b)

10
15
20
Number of generations

25

30

1.E+00

=2
Fraction of parent organism

problem encountered with some components of culture

medium is seasonal auxotrophy. This is variation in
growth and production encountered due to seasonal
change in raw materials. Often, this is not seen in the
laboratory, but appears on a large scale due to poorer
mixing in large-scale fermentors. Other considerations
for selection of nutrients are governmental regulatory
requirements or kosher certification for some foods.

1.E01

1.E02

= 0.01
= 0.0001
= 0.00001

1.E03

1.E04

10
15
20
Number of generations

25

30

Figure 1 Effect of variants on the survival of the parent strain:

(a) effect of differential growth rate (); (b) effect of variant formation
rate ().

Applied Microbiology: Industrial | Industrial Fermentation Processes

production and that maintains the seed stock under stable

conditions. Genetically engineered cultures may have
additional stability problems. Often, the genes for producing the desired product are located on plasmids that
also include selective markers, usually for antibiotic resistance. In the laboratory, these cultures can be maintained
in media that contain the selective agent. It is not practical
to use antibiotics in a large-scale plant. Therefore, production cultures must have highly stable plasmids. The
rate of plasmid loss can be much higher than variant
formation in production cultures. Even if they arent
completely lost, reduction of plasmid number can drastically reduce productivity per cell. Plasmid maintenance
usually requires a compromise between the level of
expression of the cloned genes and culture growth rate.
Increasingly, critical genes are incorporated into the
cells chromosome to minimize this problem. This also
allows the antibiotic resistance markers to be eliminated.
These markers can become regulatory, health, and environmental problems due to the potential release of
antibiotic resistance genes into the environment.

355

laboratory, culture transfers are exposed to the air, so

precautions are taken to prevent incidental contamination, such as performing transfers in rooms or hoods with
HEPA-filtered air and the wearing of sterile coveralls,
gloves, and masks by technicians. Once cultures are transferred to the plant all transfers are made through steamsterilized pipes or hoses under positive pressure.

Types of Bioreactors
Selection of a reactor design for a particular process
depends on many factors, including mass transfer considerations, mixing, shear sensitivity, broth viscosity, oxygen
demand, reliability, sterilization considerations, and the
cost of construction and operation. There are a wide
variety of bioreactor designs, generally falling into two
major categories: mechanically agitated and pneumatically agitated reactors, most commonly stirred tank and
the airlift reactors, respectively (Figures 2 and 3).
Stirred tank reactors use sparged air and submerged
impellers to aerate and mix the broth. They are designed
for highly aerobic cultures and viscous fermentations.

Large-Scale Operation
Inoculum Production
Motor

The starting place for large-scale fermentation is in the

inoculum laboratory. The fermentation is doomed to failure without pure, active inoculum, beginning with the
storage of the culture. A variety of methods are used to
store cultures under conditions that retain both genetic
stability and viability. Storing cultures in agar stabs or on
agar slants is simple and cultures can be stored at room
temperature or under refrigeration, but viability and stability are limited and cultures must be transferred
frequently, increasing the risk of contamination and
genetic changes in the culture. Lyophilization (freeze
drying) requires no refrigeration or freezing once the
cultures are preserved. It takes more skill and equipment
than slant preparation and often has significant viability
loss. Cultures are not easily revived, making it unsuitable
for use as the primary inoculum source in a plant.
Cultures can be stored almost indefinitely at room temperature and can serve as a long-term storage and as backup cultures in the event of a catastrophe. Cultures stored
in glycerol solutions at 70  C or lower generally have
high viability and are easy to produce and revive, but
require cryogenic freezers or liquid nitrogen tanks and are
thus more susceptible to power outages and equipment
failure.
Once the cultures are safely stored, they must be
revived, grown in the inoculum laboratory, and transferred to seed fermentors. Cultures are usually grown in
shake flask cultures in the laboratory and transferred to a
suitable container for transfer to the plant. In the

Gas
exhaust

Mechanical
seal

Media
fill
Steam
seal

Air
Baffle/
cooling-coil

Cooling
jacket
Impellers

Sparge
ring
Harvest
Figure 2 Stirred tank bioreactor (drawing by Wade Rambo).

356 Applied Microbiology: Industrial | Industrial Fermentation Processes

Air

Down
flow

Up-flow

Cooling
jacket

Draft
tube

Sparge
ring

Harvest
Figure 3 Airlift bioreactor (drawing by Wade Rambo).

forces are much lower, thus being useful for shearsensitive cultures or in processes where the energy cost
of agitation and heat removal is a significant factor. The
simpler mechanical design, higher reliability, and ease of
cleaning make airlift reactors more suitable for extended
or continuous operations. Most large-scale airlift fermentors are used for plant effluent treatment, production of
bakers yeast, or fungal fermentations where the size of
the mycelial pellets is controlled by shear forces.

Modes of Operation
There are three basic modes of operation: batch, fed
batch, and continuous, with variations in these three
basic modes (Figure 4). In batch mode all the ingredients
required for fermentation, with the exception of pH control chemicals (usually ammonia), are added to the
fermentor prior to inoculation. The fermentation is run
until the nutrients are exhausted; then the broth is harvested. The advantage is simplicity of operation and
reduced risk of contamination. It is useful in fermentations with high yield per unit substrate and with cultures
that can tolerate high initial substrate concentrations.
Fed batch mode starts with some of the nutrients in
the fermentor at inoculation. Concentrated nutrients are
(a)

Volume

Media
fill

Gas
exhaust

Time

Volume

Time

Volume

(c)

Time
(d)

Volume

There are many variations in design: the style, number, and

placement of impellers, the height-to-diameter ratio, and
the number and placement of coils or baffles. Thus, they
are adaptable to a variety of fermentation processes. The
drawbacks are high energy input (higher operation costs)
and mechanical complexity (increased contamination risk,
higher maintenance costs, and more difficult to clean).
They are also prone to impeller flooding, which limits air
flow, and added heat input from the agitator, which
becomes increasingly difficult to remove as the scale
increases. Despite these shortcomings, most fermentation
plants install stirred tank reactor due to their adaptability
to a variety of processes, the high risk of scale-up, and the
high capital cost of building large-scale fermentation
facilities.
Airlift fermentors mix the broth with air from the
sparger. Some designs have an internal draft tube to direct
the flow of the fluid. Most airlift designs have a greater
height-to-diameter ratio than stirred tank vessels to
improve oxygen transfer. The mixing is usually not as
good as in a stirred tank but the energy input and shear

(b)

Time
Figure 4 Changes in fermentor volume with time for different
modes of fermentor operation: (a) batch, (b) fed batch,
(c) repeated batch, (d) continuous.

Applied Microbiology: Industrial | Industrial Fermentation Processes

added as the fermentation progresses. The advantages are

the ability to add large quantities of nutrients to the
fermentor by adding them gradually and the ability to
control the rate of nutrient addition. This allows higher
product concentrations without subjecting the culture to
inhibition by high levels of nutrients. It also makes it
possible to control the culture growth rate and dissolved
oxygen (DO) concentration, which are required in some
fermentations to avoid the production of anaerobic byproducts and to maximize productivity and yield. Overall,
the use of the fermentor time is more efficient in fed batch
than straight batch fermentation, reducing fixed costs.
The disadvantages are increased risk of contamination,
due to the addition of nutrients through a continuous
sterilizer, and increased equipment costs for continuous
sterilization and flow control equipment for feed streams.
Both batch and fed batch can be run in repeated mode,
with a small portion of the previous batch left in
the fermentor for inoculum. The fresh medium is then
added through a continuous sterilizer. Use of the fermentor is increased by eliminating turnaround time, but the
risk of contamination and genetic degradation of the
culture limits the number of times a batch can be repeated
without cleaning and resterilization of the fermentor.
Usually, repeated fermentations are run for two or three
batches.
In continuous mode, the starting medium and inoculum are added to the fermentor. After the culture has
grown, the fermentor is fed nutrients and broth is withdrawn at the same rate, maintaining a constant volume of
broth in the fermentor. Ideally, the dilution rate is the
same as the culture growth rate, a condition called steady
state. Continuous mode maximizes the use of the vessel
and is especially good for fermentations that take a long
time to reach high productivity. The disadvantages are
the potential selection of adapted cultures and increased
risk of contamination, especially since it is difficult to
keep contaminants from growing back through the
continuous harvest line. As with repeated batch mode,
continuous fermentations cannot usually be run indefinitely, but fermentations of several hundred hours
duration are possible under aseptic conditions. In continuous mode with cell recycle, the cell mass is separated
from the harvested broth and returned to the fermentor.
This allows the dilution rate to be higher than the growth
rate of the culture. Fuel ethanol is often produced continuously for extended times under nonsterile conditions,
usually using a series of tanks. Pasteurized medium and
culture are added to the initial tank, with the fermentation
ending in the finishing tank with all the substrate
exhausted. Adding fresh culture on a regular basis reduces
the risk of culture selection, and contamination is kept
under control by a combination of high alcohol concentration, high yeast growth rate, and low pH.

357

Monitoring and Control

Ideally, fermentation is best controlled using online, realtime measurement of pH, oxygen, cell mass, substrate
concentration, metabolic state, and nutrient flow rates.
The data are then applied to a precise model of how the
culture will respond to changes in any of the parameters.
In the real world, only a few of the parameters are actually
measurable in real time and the models of culture behavior are often imprecise extrapolations of experimental
data, although technological advances are moving in the
direction of better monitoring and control using sophisticated modeling systems, such as artificial neural networks.
Most physical parameters temperature, pressure,
power input, impeller speed, gas, and liquid flow can
be measured accurately without invasive instrumentation.
Reliable online measurement of the chemical environment has been limited to pH, DO, and exhaust gas
analysis, but advances in infrared technology have made
it possible to measure several parameters of broth composition such as substrate, product, and ammonia
concentration simultaneously online. Various optical
density probes, to measure cell mass, and enzyme probes,
to measure nutrient or product concentration, are available. Unfortunately, these are subject to fouling or cannot
be steam sterilized. Measurement of physiological characteristics, such as intracellular ATP, DNA, and RNA
content, is not currently practical and must be inferred
from the physical and chemical data.
Most fermentations are run with a minimum of temperature and pH control. Growing microbes create a large
amount of heat, so the temperature is controlled with
chilled water circulating in a water jacket or internal
cooling coils. Consumption of carbohydrates causes the
pH to decrease, so a base, usually ammonia, is used to
control the pH in most fermentations. The ammonia also
acts as a source of nitrogen for protein production.
Fermentations using organic acids or proteins as the primary nutrient may need to have the pH controlled with
acid. In aerobic fermentations, DO concentration can be
controlled by changing the agitator speed, air flow rate, or
fermentor pressure. Fermentors that are sparged with air
or other gases produce protein foam that can be expelled
out of the exhaust vent, causing significant loss of fermentor volume, resulting in lower yield and contamination.
The foam is controlled by the addition of a liquid antifoaming agent, which is added when the foam layer comes
in contact with a conductance probe at the top of the
fermentor. Antifoam agents reduce oxygen transfer and
are toxic to many cultures, so addition must be kept to a
minimum by adding small doses with delays between
additions to allow the antifoam to mix.
In fed batch and continuous fermentation it is often
necessary to maintain nutrients at a limiting concentration in order to control the growth rate of the culture,

358 Applied Microbiology: Industrial | Industrial Fermentation Processes

maximize product yield, or avoid anaerobic conditions.

Escherichia coli fermentations are especially susceptible to
anaerobic metabolism even when enough oxygen is available because the glucose uptake rate is higher than the
rate that glucose can be processed by the tricarboxylic
acid (TCA) cycle. Under these conditions E. coli produces
acetic acid, which inhibits growth and protein production.
Substrate feed can be regulated using feedback control
by pH, DO, or exhaust gas analysis, either directly or by
calculating physiological parameters, such as substrate utilization rate or oxygen uptake rate (OUR), and by adjusting
the feed rates accordingly. In a pH-controlled feed system,
the consumption of carbohydrate drives the pH down and
a base (usually ammonia) is added to maintain a constant
pH. When the carbohydrate becomes limiting, the pH
rises. When this occurs, the carbohydrate feed rate is
increased by the control system, driving the pH down
again. Sophisticated control schemes can be devised by
measuring the rate of base consumption and the rate of
pH increase when the substrate is limiting and regulating
the feed proportionally. Measurement of pH can only
determine whether there is not enough feed, not when
there is too much, so without proper control loops in the
computer program, a pH-controlled feed can overfeed
carbohydrate, or cause the fermentation to bog down by a
cyclical decrease in response. DO-controlled fermentations
are often maintained between two set points, typically 20
40% DO, by increasing the feed when the DO rises and
decreasing the feed when it drops. Without some damping
of the response in DO-controlled fermentations there is a
tendency, at a large scale, toward excessive oscillations in
DO and feed rates due to the long mixing times. More
sophisticated control systems use a combination of the air
flow rate, dissolved oxygen and exhaust gas analysis (oxygen and carbon dioxide) to calculate the volumetric OUR
and adjust the carbohydrate feed accordingly. This has the
advantage of feeding carbohydrate based on the aerobic
capacity of the culture. In fermentations that are well
characterized and consistent, the aeration, agitation, and
feed rates can be based on a predetermined profile as long
as the fermentation is monitored for deviations and the
profile adjusted accordingly. Some fermentations require
additional feed solutions, such as salts, trace minerals, or
product precursors. These are often fed in proportion to
the carbohydrate feed or based on residual concentrations
in the fermentor broth.
Sterilization and Contamination Control
Contamination of a fermentation process can reduce productivity and yield, or completely ruin a batch. In
addition, some contaminants can be pathogenic or produce toxins that can render products unusable or be a
health risk to plant workers and consumers. Therefore,
complete asepsis is the operating philosophy of most

large-scale plants. For a plant to operate aseptically, this

philosophy must be an integral part of every aspect of the
operation, from design and construction to operating procedures and personnel training.
Sterilization is the process of eliminating all viable
organisms from equipment and materials. For a fermentation plant, this requires:
of equipment (fermentor vessels,
Presterilization
inoculation, feed and harvest piping, and air filters).
of media components, either in the fer Sterilization
mentor vessel or through a continuous sterilizer.
of aseptic conditions throughout the
Maintenance
entire fermentation process.
Some processes are relatively insensitive to contamination,
such as fermentations run at low pH, high temperatures,
with short cycle times (high growth and production rates),
or those that produce inhibitory or toxic products (alcohols,
antibiotics, and organic acids). Other processes are highly
sensitive to contamination those run at neutral pH and
moderate temperatures, employing slower-growing cultures, or with long run times. These require more
stringent measures to maintain aseptic conditions.
Most process equipment and medium components are
sterilized by saturated steam under pressure. The rate of
cell death depends on the temperature and the time of
exposure to steam, with the spores of the thermophile
Bacillus stearothermophilus used as the benchmark for determining sterilization effectiveness. The standard condition
for steam sterilization is 121  C for 15 min, but less time is
required at higher temperature and pressure. For instance,
it takes 12 min to kill 106 spores at 121  C, but only 30 s at
135  C. Other factors, such as viscosity, the concentration of
dissolved solids, and particulate matter, affect the sterilization time. The fermentation broth can be sterilized in place
or through a continuous in-line sterilizer into a presterilized
fermentor. Likewise, feed solutions can be sterilized in a
feed tank or continuously as the feed solution is added to
the fermentor. Sterilization of culture media and feed solutions in place requires a significant amount of time in a large
vessel for heating and cooling, which reduces the productive time of the fermentor and requires large amounts of
steam and cooling water. The long time required for sterilization can degrade some media components. It has the
advantage of being simple, without the need for heat
exchangers or flow control equipment, thus reducing the
risk of upsets in the process that could result in
contamination.
Continuous sterilization is sometimes used for the
fermentor broth but is more often used for nutrients
added during culture, especially for fed batch and continuous fermentations. This is usually accomplished by
direct steam injection into the nutrient stream or transfer
of heat through a heat exchanger with no direct steam
contact. After heating, a holding loop maintains the

Applied Microbiology: Industrial | Industrial Fermentation Processes

temperature for a specific time, followed by a second heat

exchanger to reduce the heat of the medium before it is
added to the fermentor. Continuous sterilization is
usually carried out at high temperature and pressure to
reduce the time. This minimizes the heat damage to the
medium and reduces the length of holding tube or
increases the flow rate through the system. Hot medium
returning from the holding loop is used to preheat the
cold medium going to the sterilizer, thus reducing the
requirements for steam to heat the incoming broth and
cooling water to cool the sterilized broth. Duplicate systems are required for sterilizers to be cleaned and
maintained properly.
Heat-sensitive solutions, air, and other gases are sterilized by using absolute membrane filters thin membranes
with pores no larger than their rated size before being added
to the fermentor. Most bacteria and spores are retained by
filters with 0.2 mm absolute pore size. Air is sterilized with
hydrophobic filters, usually preceded by prefilters that
remove particulates and water from the compressed
air and increase the life of the final sterile filters.
Bacteriophage, or phage, which are retained on dry filters
by electrostatic forces, are small enough to pass through a
0.2 mm filter if the air is wet; thus it is vital to keep the
process air as dry as possible. Microbial contamination
usually reduces the productivity and yield of a batch, but
product can often be salvaged. Phage can destroy the production culture within a few hours of introduction into the
fermentor and are easily spread throughout the plant.
Fermentation plant managers live in fear of phage.
During fermentation, the contents of the reactor must
be isolated from potential contaminating sources. Besides
filtering the air, all penetrations into the fermentor must
be sealed from the outside. Agitator shafts are sealed with
double mechanical seals that have steam between the
rotating seals. DO and pH electrodes are sealed with
O-rings or gaskets. No valves have fine enough tolerances
to prevent contamination from growing across contact
surfaces, so media fill and harvest and inoculation lines
are isolated with steam seals steam-filled sections of
pipe between two valves that connect the fermentor to
the outside environment. There is a steam source and a
relief valve to vent steam condensate and maintain steam
pressure above 15 psig when the line is not in use.
Of all the problems encountered in a fermentation plant,
contamination is the most persistent. Contamination control
is a daily, ongoing activity. The process is monitored for
contamination by microscopic examination and plating on
nutrient agar from the first shake flask through the production fermentor. This helps prevent contaminated culture
from being used in production and can be used to trace
the source of contamination when it does occur. In addition
to testing the culture, other potential sources of
contamination, such as sterile feed streams, are monitored.
Unfortunately, the volume of the sample that is practical to

359

test for contamination is very small in comparison to the

size of the process being tested, so contamination is often
not detected until it has become a serious problem.
Therefore, preventive maintenance is very important to
control contamination. Fermentors, piping, valves, and filters are routinely tested for integrity. Leaky valves and
cracks in cooling jackets or coils are common sites for
microbial contamination to enter the process, and cooling
water leaks are common sources of phage contamination.
Treatment of chilled water with oxidizing agents such as
chlorine also reduces the risk of contamination from cooling
water leaks. Proper procedures for cleaning, sterilization,
culture transfer, and fermentor operation are critical to
maintaining an aseptic process. This involves careful evaluation of the effect of procedures on process integrity,
writing procedures clearly, training fermentor operators
properly, and monitoring the effectiveness of training.
Utilities
Although fermentation processes are usually preformed at
near-ambient temperatures, energy and water consumption are high due to the need for sterilization, cooling,
agitation, and aeration as well as for downstream processing. Fermentation plants also produce a considerable
volume of waste, most requiring treatment by sewage
facilities.
Steam

Steam is required for sterilization of the medium, either in

the fermentor or through continuous sterilizers. During
the fermentation itself, a certain amount of steam is
required to maintain the microbiological integrity of the
fermentor vessel by the use of steam seals. Fermentation
plants often require large quantities of steam in downstream processing for evaporation and drying due to the
typically low concentrations of product in the broth.
Cooling water

After sterilization, fermentors need to be cooled before

they can be inoculated. During fermentation, cultures
generate metabolic heat and agitators add heat to the
broth. This must be removed with chilled water to maintain the proper incubation temperature. Chilled water is
also needed in downstream processing for crystallizers
and for condensers used to recover process water from
evaporators. Water is chilled using compressors, which
are in turn cooled with water from cooling towers. Chilled
water is usually recycled in a closed system, but there are
evaporative losses in the cooling towers. There is also a
high energy input for cooling towers and compressors.
Process water

Most of the fermentor broth is water. The quality of the

water used depends on the sensitivity of the culture to

360 Applied Microbiology: Industrial | Industrial Fermentation Processes

minerals in the water, the effect of water quality on

downstream processing, and regulatory requirements.
Food products require the use of potable water, and for
some fermentations, water must be purified by reverse
osmosis to remove excess minerals. Other fermentations
can use recycled water removed by evaporation in downstream processing. Water is also used for some
downstream process steps and for cleaning and rinsing
the fermentors. Overall water balance between chilled
water, tower water, process water, and waste streams is
critical to the operation of a fermentation plant.
Electricity

Agitators, air compressors, and chillers require a large

amount of electricity. Where economies of scale are adequate, cogeneration of steam and electricity is very costeffective. Boilers using natural gas, oil, or coal produce
very high pressure steam used for electricity generation.
The waste steam produced from generation of electricity
is at high enough pressure for use in fermentation and
downstream processing. This steam is captured and used
as the process steam for the plant, reducing the cost of
both electricity and steam. Whenever possible, the condensate water from process steam is returned to the
cogeneration plant for reuse.
Sewage treatment

Fermentation generates waste with high BOD, mainly in the

form of spent cell mass and cell by-products. Cell mass is
rich in protein and vitamins and can sometimes be used for
animal feeds. Due to regulatory restrictions on GMOs in
human and animal food, the use of cell mass from fermentations that use genetically engineered cultures in livestock
feed is not permitted in some countries. Some lower solid
streams that are generated must be treated as sewage. A high
volume of waste water can be generated, making the
throughput for treatment plants high. On-site primary and
secondary treatment can reduce high municipal sewage
charges and fines. Fermentation waste can be processed as
the primary waste treatment by anaerobic digestion, producing methane gas that can be captured and used for heating
dryers in downstream processing or electrical generation.

Common purification steps include:

and ultrafiltration to remove cell mass and
Microfiltration
other debris or retain larger molecular weight proteins.
by evaporation, centrifugation, or reverse
Concentration
osmosis.
by cooling, evaporation, or pH adjustment.
Crystallization
Distillation.
Solvent extraction.
Chromatography.

The cost of product purification varies widely, from 5 to

90% of the total production cost, and is usually proportional to the value of the product. Generally, high-value
products, such as pharmaceutical-grade proteins and
enzymes, tend to be made in lower concentration in the
fermentor and require higher product purity than bulk
chemicals or whole cell products.

Future Prospects
Concerns about global warming and diminishing stocks of
fossil hydrocarbons have encouraged research into fermentation as an alternative supply of fuels, chemicals,
and plastics from renewable resources. This is partly
responsible for the rapid increase in the US production
of fuel ethanol (Figure 5), which was expected to exceed
7.8 billion gallons in 2008. The feedstock for most ethanol
production in the United States is corn starch, and estimates of the effect of ethanol fermentation on net fossil
fuel consumption and greenhouse gas production vary
widely. These calculations depend on a number of
assumptions made about the process the energy costs
of growing corn, transporting crops, and distributing the
ethanol, as well as the effect of process by-products on the
net carbon balance. There is also concern that using food
crops for fuel will cause increases in food prices, exacerbating hunger problems in developing countries.
Producing ethanol from plant cellulose, such as corn
6000
5000

Fermentation products are often produced in dilute aqueous solutions that also include the cell mass, metabolic byproducts, and various salts, making recovery of the product
from the broth a major part of the fermentation process. The
degree of purification of the product depends on the type of
product and the end use. Industrial enzymes, for example,
are often separated from the cell mass and concentrated by
ultrafiltration to make a crude extract. At the other extreme,
antibiotics and therapeutic proteins for human use must be
very pure and require extensive downstream processing.

Millions of gallons

Downstream Processing
4000
3000
2000
1000
0
1980

1985

1990

1995
Year

2000

2005

Figure 5 Annual ethanol production in the United States.

2010

Applied Microbiology: Industrial | Industrial Fermentation Processes

stocks, grasses and wood, is projected to improve the

carbon balance of fuel ethanol and reduce the dependency on food crops. Using grasses or trees that grow on
land unsuitable for food crops may further reduce the
pressure on food supplies. There are significant hurdles
to overcome for ethanol from cellulose to become an
economically viable process. Both government and industry are devoting substantial resources toward research and
development in this area.
Another potential source of biofuels and chemicals is
algae grown in open air ponds. Algae incorporate carbon
dioxide from the air to store energy as either oil or
carbohydrate. The productivity per acre is potentially
extremely high compared to field crops, and the use of
algae that grow in salt water can reduce the dependence
on fresh water and arable land, both scarce resources. As
with ethanol production from cellulose, algae for biofuels
and chemicals will require a large investment in research
to become economical.
Replacement of plastics and other chemicals made
from petrochemicals is another focus of fermentation
research and development. Polylactic acid (PLA), a biodegradable plastic made from lactic acid, is already in
production. Other bioplastics made by fermentation are
in various stages of development, with commercial startup in the near future. Other chemical intermediate compounds may potentially be produced from organic acids
made by fermentation, such as succinic, fumaric, and
malic acids. A potential feedstock for production of
chemicals or plastics by fermentation is glycerol, a byproduct of biodiesel production.
Fermentation will continue to be used for producing
chemicals, enzymes, antibiotics, pharmaceuticals, and
foods. Increasingly, more sophisticated genetic and metabolic tools are being used to improve current processes,
develop new products, and take advantage of previously
unusable raw materials. The use of metagenomics to
sample genetic material from microbes that cannot be
cultured in the laboratory is a potential source of new
fermentation products. DNA can be sampled and amplified to explore for useful enzymes from environments
with extremes in temperature, pH, or salinity. Such
enzymes are potentially useful for industrial processes
run under harsh conditions.
Two major challenges facing traditional fermentation
processes are in conservation of energy and water, resources
that fermentation processes use in large amounts and that

361

are becoming more scarce and expensive. Recycling water,

using waste streams to generate energy, more efficient
energy use, and recapturing heat from process streams are
all ways to conserve these resources. Conservation of
resources is primarily cost driven today, but there is also
increasing political pressure to reduce greenhouse gases and
conserve other resources. Also, ongoing debates about the
use of GMOs for use in foods and concerns about the
release of genetically modified materials into the environment especially those containing genes for antibiotic
resistance limit the use of engineered cultures in some
processes.
See also: Acetic Acid Production; Amino Acid Production;
Antibiotic Production; Bioreactors; Enzymes, Industrial
(overview); Lactic Acid, Microbially Produced; Lipids,
Production; Polysaccharides, Microbial; Proteases,
Production; Solvent Production; Vitamins and Vitaminlike Compounds: Microbial Production

Further Reading
De May M, De Maeseneire S, Soetaert W, and Vandamme E (2007)
Minimizing acetate formation in E. coli fermentations. Journal of
Industrial Microbiology and Biotechnology 34: 689700.
Demain AL and Davies DE (eds.) (1999) Manual of Industrial
Microbiology and Biotechnology, 2nd edn. Washington, DC: ASM
Press.
El-Mansi EMT, Bryce CFA, Demain AL, and Allman AR (eds.) (2007)
Fermentation Microbiology and Biotechnology, 2nd edn. Boca
Raton, FL: CRC Press.
Gordon JM and Polle JEW (2007) Ultrahigh bioproductivity from algae.
Applied Microbiology and Biotechnology 76: 969975.
Junker B (2004) Scale-up methodologies for Escherichia coli and yeast
fermentation processes. Journal of Bioscience and Bioengineering
97: 347364.
Junker B, Lester M, Leporati J, et al. (2006) Sustainable reduction of
bioreactor contamination in an industrial fermentation pilot plant.
Journal of Bioscience and Bioengineering 102: 251268.
Kaur J and Sharma R (2006) Directed evolution: An approach to
engineer enzymes. Critical Reviews in Biotechnology 26: 165199.
Masato I, Ohnishi J, Hayashi M, and Mitsuhashi S (2006) A genomebased approach to create a minimally mutated Corynebacterium
glutamicum strain for efficient L-lysine production. Journal of
Industrial Microbiology and Biotechnology 33: 610615.
Patnaik R (2008) Engineering complex phenotypes in industrial strains.
Biotechnology Progress 24: 3447.
Sauer M, Porro D, Mattanovich D, and Banduardi P (2008) Microbial
production of organic acids: Expanding the markets. Trends in
Biotechnology 26: 100108.
Schmeisser C, Steele H, and Sreit WR (2007) Metagenomics,
biotechnology with non-culturable microbes. Applied Microbiology
and Biotechnology 75: 955962.