05 bacterial cultivation for production of proteins and other biological products (1)

This process includes the following steps: i preparation of the bacterial strain not described in this chapter; ii deter-mination of the growth and production parameters such as growth

1

Proteins and Other Biological Products

JOSEPH SHILOACH AND URSULA RINAS

10.1 INTRODUCTION

Large numbers of biological products are currently being

produced on an industrial scale from microorganisms such

as filamentous fungi, yeast, and bacteria These products

can be divided into several groups: primary metabolites

such as acetic acid, ethanol, and amino acids; secondary

metabolites such as antibiotics; and recombinant products,

especially proteins that are produced for pharmaceutical

purposes and technical applications In this chapter, we

concentrate on the production of biological products from

bacteria Since the basic steps of the production processes

of the various products mentioned above are similar, we

describe one of these processes in detail to give the reader

the necessary information Based on this information,

the reader will be able to design production processes for

different types of products from various types of

microorgan-isms The process we describe in detail is the production of

recombinant proteins from Escherichia coli (34, 36) This

process includes the following steps: (i) preparation of the

bacterial strain (not described in this chapter); (ii)

deter-mination of the growth and production parameters such

as growth strategy, production strategy, medium

composi-tion, pH, and optimal concentration of dissolved oxygen

(DO) and temperature (not described in this chapter); (iii)

preparation of the growth vessels; (iv) preparation of the

starter culture (inoculum); (v) bacterial growth and product

formation; (vi) process termination and preparation for the

protein recovery step; and (vii) protein recovery and

purifi-cation (not described in this chapter)

PRODUCTION FROM E COLI

10.2.1 General Process Description

Following the determination of the batch production size,

the proper growth vessels are prepared: this includes the

bioreactors used for growth of the starter culture (in some

cases, the volume required is large and more than one

trans-fer is needed) and for production The preparation involves

(i) making the culture medium for growth and production,

(ii) electrode calibration and installation, (iii) assembly

of hoses to transfer the culture from one growth vessel

to another (in cases where there is no permanent pipe

connection between the various growth vessels), and

(iv) sterilization of the bioreactor In addition, there is the need to prepare and/or calibrate various follow-up instru-ments such as pH meter, spectrophotometer, conductivity meter, glucose analyzer, and microscope The recombinant bacteria are removed from storage (working cell bank) and transferred to the starter culture shake flask or bioreac-tor containing the proper medium and are grown to the required density (which was determined when the process was developed) In some cases, cells are first transferred to

an agar plate and a single colony is used to inoculate the starter culture; in other cases, freshly transformed cells are needed for inoculation of the starter culture When the starter culture reaches its designated density, it is transferred

to the production bioreactor and then the production process commences In most cases, the production process involves two phases, i.e., a growth phase and a production phase In the growth phase, the bacteria are grown to a high density by implementing a fed-batch growth procedure (see section 10.2.6.3) The second phase is associated with the production of the recombinant protein by inducing its biosynthesis In some cases, the growth conditions after the induction are different from those before the induction Af-ter the bacAf-teria have synthesized the desired recombinant protein to the expected level, the culture is cooled down and the cells (if the product is accumulated in the cells)

or the supernatant fluid (if the product is secreted into the outside medium) are collected and processed The process flow is summarized in Fig 1

To give the reader a better feeling for the general process described above, we provide here details on the production

process of recombinant Pseudomonas aeruginosa exotoxin A

in E coli (5), which has been adapted for routine

produc-tion For a batch production size of 50 liters, a bioreactor with 50 liters of medium is prepared and sterilized One liter of starter culture is prepared by inoculating a 2.8-liter Fernbach flask containing 1 liter of medium with a frozen culture stock After 12 h of growth at 37°C, the starter culture is transferred to the bioreactor and the bacteria are grown at 37°C at a 30% DO concentration and a pH of 6.8 When the culture’s optical density reaches the value

of 40 (measured at 600 nm), the inducer thiogalactopyranoside (IPTG) is added to a final concentra-tion of 100 mmol liter1 and growth is continued for 30 to

60 min; the culture is then cooled down and the cells are collected and processed

10

10.2.2 Instrumentation and Infrastructure Required

To conduct a process of recombinant protein production

with E coli, the investigator needs to have access to the

following instrumentation

1 Cold storage equipment: a 80°C freezer to store the

bacterial master and working cell banks; a 20°C freezer

to store collected samples; a 4°C cold room; and 4°C

refrigerators to store samples, medium, agar plates, etc

2 Sterilization equipment: a steam-operated autoclave

that can hold a benchtop stirred-tank bioreactor in a

volume up to 10 liters

3 Propagation equipment: incubators in the range of 20 to

45°C to grow the bacteria on agar plates; incubator

shak-ers that can accommodate different sizes of shake flasks,

from 50-ml Erlenmeyer flasks to 2.8-liter Fernbach

flasks, which are needed for the initial growth of the

culture from the plates; and stirred-tank bioreactors in

various sizes to be used for both starter culture growth

and protein production Depending on their size, these

bioreactors can be divided into two types: one group

includes bioreactors up to 10 liters These are called

benchtop reactors and, in most cases, are sterilized in

the autoclave The other group includes bioreactors with

higher volumes that are sterilized-in-place In most cases,

the bioreactors used for bacterial growth are stirred-tank

reactors equipped with air inlet, air outlet, impellers,

baffles, air sparger, and numerous inlets and outlets for

removal of samples and medium and for adding various

solutions to the growing culture These include nutrients and growth factors to support growth and production, acid or base for pH control, and antifoam for foam control The bioreactor is also equipped with openings for installation of various probes, especially for pH and

DO A general scheme of the stirred-tank bioreactor can

be seen in Fig 2 To add various solutions to the bio-reactor, it should also be equipped with variable-speed pumps, either sterilizable or outfitted with sterilizable tubing The bioreactor is supported by instrumentation

to measure and control agitation, airflow, temperature, pressure, pH, DO, and foam In some cases, it can also include instruments to analyze the concentrations of

CO2 and O2 in the off-gas The measurements of all these variables are collected by a digital control unit that can also be used for process control based on the analysis of one or more process variables The bioreactor

is connected to a source of water, air, oxygen, and steam and also to a drain Since during the process foam can be generated by the growing culture, the bioreactor should

be equipped with a foam probe that detects the foam level and triggers the addition of antifoam solution (see section 10.2.4) Another instrument is a level probe that can detect the liquid level in the bioreactor General information on bioreactor principles and operation can

be found in several comprehensive books (3, 15, 32)

4 Analytical instrumentation: to control and follow bacterial growth and protein production, access is needed to the following analytical instruments: (i) optical microscope

FIGURE 1 General layout of the bacterial cultivation process for production of proteins and

other biological products

to check the condition of the bacterial culture; (ii)

spec-trophotometer for the measurement of bacterial density;

(iii) pH meter; (iv) benchtop centrifuge to separate the

bacterial mass from the supernatant fluid; and (v)

addi-tional instrumentation required for product measurements,

such as gel electrophoresis, enzyme-linked immunosorbent

assay apparatus, and in some cases, equipment to measure

the composition of the off-gas (e.g., mass spectrometer)

5 Processing equipment: equipment is needed to separate

the bacterial biomass from the medium The separation

can be done with a continuous centrifuge or a tangential

flow device (4)

10.2.3 Seed Culture: Preparation and Storage

Following the research and development stage, a bacterial

producer strain or strains are selected These strains are

usually stored frozen at 80°C or stored as freeze-dried

samples (lyophilized) The currently accepted procedure

is to grow the selected strains in their specified medium

and to collect the cells at the mid-logarithmic phase Once

collected, the cells are prepared for storage The cells (e.g.,

E coli) are suspended in an equal volume of a special

freez-ing medium with the followfreez-ing composition: 6.3 g K2HPO4,

1.8 g KH2PO4, 0.45 g sodium citrate, 0.09 g MgSO4, 0.9 g

(NH4)2SO4, 44 g glycerol, and 450 ml water The

sus-pended cells are divided into 0.5-ml aliquots in cryogenic

tubes and stored in the −80°C freezer Preparation of

freeze-dried aliquots of other bacteria requires special equipment

and is not described in this chapter Detailed descriptions

of preservation methods for different bacteria can be found

in ATCC Preservation Methods (30) and in Maintenance of

Microorganisms (11) The aliquots prepared from the grown

culture are kept as the master cell bank An aliquot from the master cell bank is than used to prepare a working cell bank in the same way When there is a need to start a production process, a sample from the working cell bank is taken out and used for inoculum preparation that later will

be used for the production process

10.2.4 General Information on Medium Composition, Preparation, and Sterilization

Chemotrophic bacteria need various chemical compounds designated as substrates for cell maintenance, cell growth, and production Most bacteria used for the production of low- and high-molecular-weight organic compounds (e.g., acids, DNA, proteins) are chemoorganotrophic; thus, in addition to inorganic substrates, they also need organic substrates for cell maintenance, growth, and production The substrates required by these bacteria can be grouped into two categories: (i) substrates that serve as an energy source and as a building unit to generate more cells or product, and (ii) substrates that only serve as building units for generating more cells and product, but their transformation by the bacterial cells does not generate energy

Carbon substrates such as glucose, glycerol, and other compounds containing carbon atoms can be used by these bacteria for the generation of biomass and product and for

FIGURE 2 General scheme of a stirred-tank bioreactor.

the generation of energy through substrate-level

phosphory-lation, and during complete oxidation using the respiratory

pathway Other substrates, such as salts containing nitrogen,

phosphor, and sulfur atoms, only serve as building units for

generating more cells and/or product For example, the

el-emental composition of E coli grown on a defined medium

is CH1.85O0.574N0.22 plus 12% ash (10) This elemental

composition does not vary significantly with the growth

rate In addition to these elements, cells need phosphorus

for the formation of RNA and DNA, and sulfur for the

for-mation of the amino acids methionine and cysteine Other

trace elements, such as metal ions, are required by various

enzymes for their proper function Some metal ions are

re-quired in high concentrations, such as iron needed for the

heme-containing enzymes of the respiratory chain Other

trace metals are required in small amounts, such as copper,

as there are few copper-containing enzymes In some cases,

bacteria have specific requirements for compounds that

they cannot synthesize; these compounds must be added

to the medium in order to allow cell growth An example

is the protein producer E coli K-12 strain TG1, which is a

thiamine auxotroph and therefore requires

supplementa-tion with thiamine when grown on a chemically defined

medium (10, 12)

The composition of a defined medium that can support

both small-scale (test tube or shake flask) and large-scale

batch cultivations of E coli is described in Table 1.

Preparation of the Medium

1 Dissolve KH2PO4, (NH4)2HPO4, and citric acid in

800 ml of deionized water in a beaker

2 Add trace element solutions

3 Adjust the pH of this solution to 6.8 using 5 mol liter1

NaOH and fill it up to 900 ml using deionized water

Transfer to a 1-liter bottle

4 Add MgSO4 into a 50-ml bottle and fill up to 20 ml

5 Add glucose into a 100-ml bottle and fill up to 80 ml

6 Sterilize these three bottles in an autoclave for 30 min at 120°C

7 Mix all three components under sterile conditions You can store the sterile medium for approximately 4 weeks

at room temperature

8 Add the volume of medium you need to sterile flasks

9 If necessary, add filter-sterilized thiamine (4.5 mg liter1) and antibiotics

When this medium is used in larger-scale bioreactors,

KH2PO4, (NH4)2HPO4, and citric acid are added directly

to the bioreactor and heat-sterilized The other solutions are prepared in concentrated form in separate containers

It is important to note that some compounds should be heat-sterilized separately For example, magnesium and phosphate should not be heat-sterilized together as they will form a precipitate that will not dissolve again Also, glucose should be heat-sterilized separately, as it forms brown Mail-lard products when heat-sterilized with other compounds, e.g., amino acids Another group of medium components are heat-labile and therefore need to be filter-sterilized Most antibiotics and compounds such as thiamine and IPTG, a common inducer used to initiate recombinant protein production, are not heat-sterilized and need to be filter-sterilized through a 0.22-μm-pore-size filter

For growth of E coli to high cell densities, it is

impor-tant to supply the required substrates in such a way that their concentrations are below growth-inhibitory values in the bioreactor For example, cells will not grow when the glucose concentration exceeds 50 g liter1, the phosphate concentration 10 g liter1, and the ammonium concentra-tion 4 g liter1 On the other hand, magnesium phosphate has a very low solubility, and therefore for high-cell-density cultivations it is recommended to add the majority of

TABLE 1 Defined medium using glucose as carbon substratea

A Components of medium

Component Concn (g liter1) In vol of H2O Glucose·H 2 O 12.00 80 ml

MgSO 4 ·7H 2 O 1.20 20 ml

KH 2 PO 4 13.30

(NH 4 ) 2 HPO 4 4.00 900 ml

Citric acid·H 2 O 1.70

B Trace elements

Trace element Concn (mg liter1)

Concn in stock solution (mg ml1) Add vol (ml) Fe(III)citrate 100.80 12.00 8.40

CoCl2·6H2O 2.50 2.50 1.00

MnCl2·4H2O 15.00 15.00 1.00

CuCl2·2H2O 1.50 1.50 1.00

Na2MoO4·2H2O 2.10 2.50 0.84

Zn(CH 3 COOH) 2 ·2H 2 O 33.80 13.00 2.60

Titriplex III (EDTA) 14.10 8.40 1.68

a

º

»

¼

needed phosphate at the beginning of the cultivation and

the needed magnesium continuously during the cultivation

(12, 27) Nitrogen addition to high-cell-density cultures is

done in a similar way, since adding all the required nitrogen

at the beginning will inhibit growth The nitrogen is added

continuously to the growing culture as ammonium

hydrox-ide in response to the change of the pH An example of

defined medium used for growing nonrecombinant E coli to

high cell densities of 128 g liter1 dry cell mass using glucose

(12) and 165 g liter1 dry cell mass using glycerol as carbon

source (23), and for production of recombinant proteins

(1, 7, 27), is given in Table 2 This medium has also been

successfully applied to produce 27.5 g liter1

amorpha-4,11-diene, a precursor of the antimalarial drug artemisinin, with

a genetically engineered strain of E coli in high-cell-density

culture (37) The feeding solution of this medium can be

adapted for usage in carbon-limited continuous culture

experiments by decreasing the phosphor and increasing the

nitrogen content (10)

10.2.5 Starter Culture Preparation

The starter culture (inoculum) preparation for the

recom-binant protein production process can be divided into the

following steps (i) Choose the proper growth vessel to

accommodate production batch size The starter culture

volume is usually between 0.25 and 1% of the initial

pro-duction volume For starter culture volumes of up to 4 to

5 liters, shake flasks are sufficient, but for higher volumes,

it is better to grow the starter culture in a bioreactor (ii)

Prepare the medium and the growth vessels; for details

refer to section 10.2.6.1 (iii) Inoculation of the starter

culture is usually done in two phases In the first phase,

an aliquot of the working cell bank is removed from

the −80°C freezer and transferred to a small shake flask,

containing usually between 50 and 100 ml of medium

In some cases, the first culture will be inoculated from a

single colony of freshly transformed cells or from a single

colony of a fresh agar plate generated from the

work-ing cell bank In the second phase, this culture, when it

reaches the desired density, is used to inoculate the starter

culture vessel or vessels

10.2.6 Growth and Production

10.2.6.1 Bioreactor Preparation

The bioreactor preparation process can be divided into the following steps (i) Making sure that the bioreactor is clean, that it is equipped with an inlet and an outlet air filter in good shape, and that all the valves controlling the addition ports, the sampling ports, and the harvest port are working

satisfactorily (ii) Installing the on-line probes and

calibrat-ing them In most cases, the only probes required are those for pH and DO (iii) Medium preparation: the medium

is usually composed of heat-stable and heat-sensitive re-agents (see section 10.2.4) The heat-stable rere-agents can be sterilized directly in the bioreactor and the heat-sensitive reagents are filter-sterilized as concentrated solutions in a separate container that can be connected aseptically to the bioreactor Sometimes, it is not possible to heat-sterilize certain reagents together, and there is a need to separately prepare concentrated solutions of those heat-stable in-gredients and to heat-sterilize them in the autoclave in a separate container that can be aseptically connected to the bioreactor following the sterilization

As indicated above, there are two types of bioreactors: those that are sterilized in the autoclave (not more than

10 liters in working volume) and those that are sterilized-in-place (above 10 liters) To sterilize the bioreactor in the autoclave, it is important to ensure that the air outlet is open and that all the inlet or outlet ports (which are submersed

in the growth medium) are either plugged or connected to

a port that is not submersed The air inlet filter is usually sterilized separately and is hooked to the sparger port after sterilization A 10-liter bioreactor should be sterilized for

an hour Following the sterilization, the bioreactor is placed next to its controlling instruments, the air source is con-nected to the inlet of the air filter, and the outlet of the air filter is connected to the sparger The bioreactor is allowed

to cool to the growth temperature and is ready for inocula-tion When dealing with sterilized-in-place bioreactors, the sterilization process has several steps that are coordinated and monitored, in most cases, by a programmed controller The following is a description of the process (i) Agitation

TABLE 2 Defined medium to grow E coli to high cell density using fed-batch culture technique or to grow E coli in

continuous culture

Medium components Batch culture Feeding solution,

fed-batch culture

Feeding solution, continuous culture Glucose·H2O 27.5 g liter 1 875 0 g liter 1 11.0 g liter 1

KH2PO4 13.3 g liter 1 2.7 g liter 1

(NH4)2HPO4 4.0 g liter 1 0.8 g liter 1

MgSO 4 ·7H 2 O 1.2 g liter1 20.0 g liter1 1.0 g liter1

Citric acid·H 2 O 1.7 g liter1 0.35 g liter1

Fe(III) citrate 100.8 mg liter1 40.0 mg liter1 12.0 mg liter1

CoCl 2 ·6H 2 O 2.5 mg liter1 4.0 mg liter1 0.5 mg liter1

MnCl 2 ·4H 2 O 15.0 mg liter1 23.5 mg liter1 3.0 mg liter1

CuCl 2 ·2H 2 O 1.5 mg liter1 2.3 mg liter1 0.3 mg liter1

H 3 BO 3 3.0 mg liter1 4.7 mg liter1 0.6 mg liter1

Na 2 MoO 4 ·2H 2 O 2.1 mg liter1 4.0 mg liter1 0.5 mg liter1

Zn(CH 3 COOH) 2 ·2H 2 O 33.8 mg liter1 16.0 mg liter1 1.6 mg liter1

Titriplex III (EDTA) 14.1 mg liter1 813.0 mg liter1 1.7 mg liter1

is turned on, and the bioreactor is heated up by steam that

flows into the bioreactor jacket (ii) When the temperature

reaches 100°C, the steam is allowed to go directly into the

medium through the air inlet filter, sterilizing the air filter at

this time and raising the medium temperature to 121.5°C

(iii) At this point, it is advisable to sterilize all the auxiliary

ports Each port is equipped with its own steam inlet and

condensate outlet The bioreactor is kept at this temperature

for at least 20 min and is allowed to cool down to the growth

temperature by circulating cold water through the bioreactor

jacket (iv) It is essential to confirm that when the medium

temperature reaches below 100°C, air can flow into the

bio-reactor to compensate for pressure loss due to condensation

(v) When the vessel reaches the growth temperature, it is

ready for inoculation

Specific details of the recombinant P aeruginosa

exo-toxin A production process adapted to routine production

are based on procedures described before (5)

Preparation of a Bioreactor Containing a 50-Liter

Working Volume

1 DO and pH electrodes are installed; the bioreactor

is filled with 45 liters of distilled water containing

250 g of yeast extract (Difco), 500 g of tryptone

(Difco), 250 g of NaCl, 250 g of K2HPO4, and 5 ml of

antifoam P-2000 (Fluka)

2 The bioreactor is then heat-sterilized Three

solu-tions are heat-sterilized separately: (i) 4 liters of 50%

glucose solution in a 5-liter transferring bottle, (ii)

a 500-ml solution of 123.24 g (1 mol) MgSO4·7H2O

in a bottle, and (iii) 50 ml of trace element solution

(27.0 g liter1 FeCl3·6H2O, 2.0 g liter−1 ZnCl2·4H2O,

2.0 g liter1 CoCl2·6H2O, 2.0 g liter−1 Na2MoO4·2H2O,

1.0 g liter1 CaCl2·2H2O, 1.0 g liter−1 CuCl2, 0.5 g

liter−1 H3BO3, 100 ml liter−1 concentrated HCl) In

addition, a 500-ml solution of 5 g ampicillin is

filter-sterilized

3 Following the sterilization of the bioreactor, the above

solutions (glucose, MgSO4, trace element solution, and

ampicillin) are added into the bioreactor

4 In addition, a solution of 50% ammonium hydroxide (approximately 2 liters) for pH control is prepared in an aspirator bottle equipped with silicone tubing suitable for peristaltic pumping and connected to the bioreactor At this point, the bioreactor is ready for the inoculation

10.2.6.2 Inoculation

The volume and density of the starter culture (inoculum) depend on the process development parameters and on the volume of the production bioreactor In general, the starter culture should be in the middle of the logarithmic growth phase, and the volume can be somewhere between 0.25 and 1% of the initial production volume After verifying that the starter culture is not contaminated (using a light microscope) and that it is in its proper growth phase, it is transferred aseptically to the production vessel To ensure that the starter culture is not contaminated, it is advisable

to streak an agar plate for later visual colony inspection If the starter culture grew in another bioreactor, it is trans-ferred directly from that bioreactor to the production bio-reactor via a sterilized hose using a pump or by pressurizing the starter culture bioreactor If the starter culture is grown

in shake flasks, it is transferred first to a transfer container and from this container into the production bioreactor by either pressure or pump

The details associated with recombinant P aeruginosa

exotoxin A production are as follows (i) One liter inocu-lum is grown for 12 h at 37°C in the following medium: 5

g liter1 yeast extract, 10 g liter1 peptone, and 5 g liter1 NaCl (ii) After overnight growth, the pH and the optical density (OD) are analyzed, and if they are in the accepted limits, the culture is transferred to the bioreactor to start the growth and production process

10.2.6.3 Growth Strategies

There are three major strategies to grow bacteria—batch, fed-batch, and continuous culture—shown schematically in Fig 3 When cells are grown in a batch procedure, all nutri-ents are added at the beginning of the cultivation, and the cell growth and production process ends when the essential nutrients are depleted The limiting essential nutrient, in

FIGURE 3 General scheme of cultivation strategies.

most cases, is the carbon source, and in the E coli growth

process it is usually glucose Since most E coli strains are

sensitive to high glucose concentrations, the final cell

density and the final product concentration are relatively

low E coli strain B is exceptional in its capability to

toler-ate glucose concentrations as high as 40 g liter1 without

excessive acetate formation and therefore can grow to a

relatively high density and consequently produce a higher

level of product while growing in a batch mode strategy

(21) The batch culture technique is simple to implement

and can be handled in laboratories that cannot

accom-modate sophisticated growth strategies For recombinant

protein production in batch culture, it is recommended

to use either a complex medium, e.g., Luria broth (LB) or

terrific broth, supplemented with either glucose or glycerol

as an additional carbon substrate or a defined medium as

described in section 10.2.4 With the defined medium

de-scribed in section 10.2.4, cell densities of approximately 10

g liter1 dry cell mass (corresponding to an optical density

of approximately 20 at 600 nm) can be obtained in batch

culture for E coli K-12 strain TG1 at 20 g liter1 glucose

(12) The maximum protein concentration that can be

reached is affected by the properties of the protein and the

expression vector used for production

In order to obtain higher productivities, fed-batch

cul-ture strategies are being used In these growth strategies,

one of the essential growth components, usually the carbon

source, is added continuously to the growing culture As

stated above, in batch cultivations the final cell

concen-tration is limited by the initial glucose concenconcen-tration

However, high glucose concentrations usually cause acetate

formation, which will decrease the biomass yield on glucose

or even completely inhibit bacterial growth (28, 29)

Fed-batch cultivation eliminates acetate formation by adding

the glucose continuously into the bioreactor but keeping

its concentration below a detectable level When using

this growth strategy, it is important that all other nutrients

are in excess, so that the growth is controlled only by the

available carbon source To allow growth at a defined but

restricted growth rate under carbon-limiting conditions,

the glucose (or another carbon substrate such as glycerol) is

added to the bioreactor as follows (12):

ms(t)  F(t)SF(t)  ( M(t)

YX/S  m ) V(t)X(t) (1)

where ms is the mass flow of substrate (g h1), F is the

volumetric feeding rate (liters h1), SF is the concentration

of the substrate in the feeding solution (g liter1), μ is the

specific growth rate (h1), YX/S is the biomass/substrate

yield coefficient (g g1), m is the specific maintenance

coefficient (g g1 h1), X is the biomass concentration

(g liter1), and V is the cultivation volume (liters) In a

fed-batch system, the following growth equation applies:

d(XV)

Assuming M (growth rate) does not change with time,

one obtains on integration of equation 2, when starting the

feeding at time tF:

X(t) V(t)  X t F V t F eM(t  t F ) (3)

Thus, by introducing equation 3 into equation 1, the

substrate mass feeding rate for a constant specific growth

rate (Mset) follows as

ms(t)  ( Mset

YX/S  m ) V t F X t F eMset(t  t F ) (4)

To allow E coli to grow to high cell densities, a growth

rate must be chosen (Mset) that does not lead to the forma-tion of acetic acid It is generally the case when the specific growth rate μ is below 0.15 h1 This feeding strategy allows exponential growth at a constant specific growth rate when the yield coefficient YX/S and the maintenance coefficient m

do not change with time It implies that the same amount

of biomass is generated per amount of carbon consumed and that the cells always use the same amount of carbon per bio-mass and time unit for maintaining vital cell functions This exponential feeding strategy, called “predetermined feeding protocol,” does not depend on the measurement of any growth variables There is no need to continuously measure the bacterial concentration, the oxygen consumption, or the carbon dioxide production It is only required to know the time for starting or changing the feeding, the culture volume and the biomass concentration at these time points, and the yield and maintenance coefficients, and to choose the desired growth rate (Mset) As a rough assumption, a yield coefficient YX/S of 0.5 and 0.45 for glucose and glycerol, respectively, and a maintenance coefficient of m  0.025 g

g1 h1 for both substrates can be considered If a desired specific growth rate (Mset) of 0.12 h1 is chosen, formation

of acetic acid should not be observed For temperature-induced production of recombinant proteins, the desired specific growth temperature should be reduced to 0.08 h−1 after raising the temperature to 42°C (1, 25, 27)

Fed-batch cultivations are normally preceded by batch culture growth, and the fed-batch phase of the cultiva-tion is started when the glucose of the batch phase is consumed This can be followed by monitoring the DO concentration, which will sharply rise after all glucose has been consumed It is possible to start the feeding according to equation 4 after this rise in the DO concen-tration Another option is to wait until the acetate that accumulated in the batch phase has been consumed by the cells This can also be followed by monitoring the DO concentration After the sharp rise in the DO concentra-tion as a result of glucose depleconcentra-tion, the bacteria will start

to consume the accumulated acetate; this will be indicated

by a slow decline of the DO concentration When the DO concentration rises again, all acetate has been consumed

by the cells and feeding can be started

The fed-batch procedure described above is a simple feed-forward strategy allowing exponential growth at a con-stant specific growth rate as long as the carbon source yield and maintenance coefficients do not change with time, as assumed in equations 1 to 4 In cases when programmable pumps are not available, it is possible to manually adjust the carbon source feeding rate by stepwise increases in such a way that it follows a pseudoexponential increase as prede-termined by that same equation

Another, simpler fed-batch strategy involves linear feed-ing In this case, the amount of glucose or any other carbon source added per unit time does not vary with the culture time A drawback of this linear feeding strategy is that it leads

to declining growth rates with the increase in biomass In some cases, mixed fed-batch strategies are applied First, an exponential feeding strategy is implemented to allow growth

at a constant specific growth rate The culture is grown in this way until the supply of DO reaches its limitation At this point, the feeding is switched to linear feeding Fed-batch cultivation strategies can also be based on the metabolic ac-tivities of the growing culture, such as oxygen consumption or

pH changes For example, the change in the DO concentra-tion activates a pump that delivers the carbon source in such

a way that a certain DO concentration is maintained (17)

More-detailed discussions on the pros and cons of different

fed-batch strategies can be found elsewhere (13, 22)

Fed-batch processes are the most common strategies

in industrial settings However, in some cases, continuous

culture techniques are used In continuous culture the

carbon source is added to the growing culture at a certain

rate, but unlike the fed-batch technique, where the culture

volume increases with time, in the continuous culture the

total volume of the culture is kept constant and the excess

culture volume, containing cells and product, is collected

at the same rate Theoretically, the highest productivities

are reachable in continuous cultures, provided the bacteria

exhibit sufficient genetic and physiological stability (16) In

most cases, high-producer strains designed by genetic

engi-neering or traditional mutagenesis will lose their production

capabilities in long-term continuous cultures

10.2.6.4 Following Growth and Production

The growth and production process starts after the

bio-reactor has been inoculated with the starter culture The

follow-up of the process is done by monitoring both on-line

and off-line data The common on-line data are DO

con-centration, pH, agitation (revolutions per minute), airflow

(liters per minute), temperature, bioreactor pressure, and

the accumulative volume of acid or base added to keep the

pH at its predetermined value In some cases, other on-line

data are monitored, such as the CO2 and O2 concentrations

in the outlet air, and the turbidity The DO concentration

and the pH are important variables that have direct effects

on the growth and production process and therefore must be

continuously monitored and controlled In most protein

pro-duction processes by recombinant E coli, the DO

concentra-tion is kept around 20 to 30% air saturaconcentra-tion by varying the

agitation, airflow, and pressure independently, sequentially,

simultaneously, or based on a specific control strategy The

pH is controlled usually at around 7 by the addition of acid

or base depending on the medium In the case of an E coli

recombinant protein production process, the carbon source

is usually glucose and the pH is controlled by the addition

of ammonium hydroxide, and seldom by the addition of sodium hydroxide As was mentioned in section 10.2.1, this process has usually two phases: in the first phase the cells are grown to the desired density, and in the second phase the recombinant protein production is induced An example of

pH and DO control together with the measurement of other

on-line variables during an E coli protein production run is

shown in Fig 4 It is important to note that a specific control algorithm is implemented to keep the DO at 30% saturation

by increasing the agitation and the airflow with time In ad-dition, base is added to keep the pH at a value of 7

The off-line data are measured using a sample removed from the bioreactor through a special sampling port These data include the bacterial concentration, concentrations

of various substrates such as glucose or metabolites such

as acetic acid, and the product concentration Bacterial concentration is usually evaluated by measuring the turbid-ity of the culture using a spectrophotometer This method provides quick information on the bacterial concentration when the medium is clear If the medium is not clear, it is not possible to assess the bacterial concentration by turbid-ity measurement, and in such cases, the packed cell volume can be an alternative Other methods, such as dry cell mass measurements and cell counting, are time-consuming and

do not provide data in real time Glucose concentration can

be measured by high-pressure liquid chromatography or by

a glucose analyzer based on the enzyme glucose oxidase In most cases, the amount of the product cannot be determined during the production process itself due to the time required for analysis and therefore is done later on stored samples

An example of measuring off-line bacterial concentration

by OD at 600 nm and glucose concentration by glucose ana-lyzer (YSI Inc., Yellow Springs, OH) is shown in Fig 5

Additional details of the process for recombinant P

aeruginosa exotoxin A production are listed here The fol-lowing on-line variables are monitored during the process:

DO concentration, pH, airflow, agitation, pressure, amount

of base added, and temperature (Fig 4) Throughout the pro-cess, the DO concentration is kept at 30% air saturation and

FIGURE 4 On-line data on batch cultivation process for production of recombinant exotoxin A

from E coli in 5-liter bioreactor Arrow A indicates the point of introducing oxygen-enriched air to

the culture; arrow B indicates the time when IPTG was added to the culture

is controlled by simultaneously increasing the agitation and

the airflow The pH is kept at 6.8 by adding 50% ammonium

hydroxide solution automatically, and the amount of the base

added is monitored continuously The following variables are

measured off-line: bacterial concentration is determined by

the OD value at 600 nm and the glucose concentration is

measured using the glucose analyzer made by YSI (Fig 5)

E coli BL21, the strain used for the production, can tolerate

glucose concentrations as high as 40 g liter1 (21); therefore,

the cultivation is carried out as a batch process

10.2.6.5 Bioprocess Calculations

Growth of most bacteria, including E coli, occurs by cell

division Thus, during unlimited growth (as found in the

batch phase when all nutrients are in excess), growth can

be described as follows:

dX _

or in the integrated form:

X  X0eM(t  t0) (6)

where X is the biomass (g) at time t (h), X0 the biomass

at t0 (usually the beginning of the cultivation; t0  0 h),

M the specific growth rate (h1), and e the Euler number

(2.718281828 .)

The specific growth rate can be determined from

mea-surements of the bacterial biomass (e.g., analysis of the

opti-cal density) at different time points as follows:

M  lnX  lnX t  t 0

During carbon-limited fed-batch cultivation when no

acids are produced, the growth of bacterial cells can also be

determined on-line by following the ammonia consumption

(26) Thus, when ammonia is used for pH control, it does

not only serve as a base for pH control but also as a

nitro-gen source In contrast to the biomass yield coefficient for

carbon substrates such as glucose, YX/ C H O , which is not

constant, the biomass yield coefficient for nitrogen, such

as ammonia, YX/N H 3 , is constant and is not affected by the metabolic status of the cells

When YX/N H 3 (g g1) and the concentration of ammonia

in the feeding/base solution are constant, their absolute val-ues are not required and the actual specific growth rate, M, (h1), can be calculated from the time-dependent change

of the natural logarithm of the dimensionless signal of the ammonia balance, MN H 3 (g g1), according to equation 8

M  _ dln(M NH 3 )

The actual biomass in the bioreactor, X (g), can be cal-culated according to equation 9:

X  MN H 3 CN H 3 YX/N H 3 (9)

where MN H 3 (g) is the mass of the ammonia solution added into the bioreactor (g), CN H 3 is the concentration of ammo-nia in this solution (g g1), and YX/N H 3 is the average bio-mass yield coefficient with respect to ammonia (7 g g1) The volumetric oxygen and carbon dioxide transfer rates (OTR and CTR, respectively) (g liter1 h1) can be calcu-lated from the mass balance of the gas phase as follows (10): OTR  M O 2 F G

in _

V(t)VM ( x O

2

in (t)  x O

2

out (t)

1  x O

2

in (t)  x CO

2

in (t)

1  x O

2

out (t)  x CO

2

out (t) ) (10)

and CTR  M CO 2 F G

in _

V(t)VM ( x CO

2

out (t)

1  x O

2

in (t)  x CO

2

in (t) _

1  x O

2

out (t)  x CO

2

out (t)  x CO

2

in (t) ) (11)

where M O 2 and M CO 2 are the molecular mass of oxygen and carbon dioxide (g mol1), respectively; F Gin is the volumetric inlet airflow (liters h1) at standard conditions;

V(t) is the working volume of the bioreactor (liters); VM is the mol volume of the ideal gas (liters mol1) at standard conditions; x in and x in are the molar fractions of oxygen

FIGURE 5 Off-line data on batch cultivation process for production of recombinant exotoxin A

from E coli in 5-liter bioreactor The arrow indicates the time when IPTG was added to the culture.

and carbon dioxide (mol mol1), respectively, in the inlet

air; and x O out2 (t) and x CO out2 (t) are the molar fractions of

oxy-gen and carbon dioxide (mol mol1), respectively, in the

outlet air of the bioreactor For calculation of specific rates,

the convective flow of oxygen and carbon dioxide can be

neglected and the transfer rates OTR and CTR can be

con-sidered to be identical to the oxygen uptake and carbon

di-oxide evolution rates Specific rates can then be calculated

by dividing volumetric rates by cell concentration

10.2.6.6 Initial Product Recovery

Depending on the process and the product, the

recom-binant protein can accumulate inside the cells or can be

secreted into the growth medium When the product is

secreted into the medium, the bacterial biomass is separated

from the medium by either centrifugation or filtration and

the protein is recovered from the supernatant When the

product accumulates in the cells, it is possible to recover the

protein either from the biomass after its separation from the

medium or, especially when the cell concentration is high,

directly from the complete broth containing both the cells

and the growth medium without further separation Details

on continuous-flow centrifuges and tangential filtration

systems can be found in several books dealing with

down-stream processing and protein purification (4, 33)

For example, the following steps are required for the

downstream processing of P aeruginosa exotoxin A after

termination of its production in the bioreactor The protein

is secreted into the periplasmic space, thus accumulating

inside the cells The initial downstream processing involves

the separation of the cells from the medium, followed by

lysis of the cells by osmotic shock, which releases the

ac-cumulated protein into the supernatant The recovery and

clarification of the supernatant and final purification of the

protein are described in detail elsewhere (8)

10.3 OTHER PRODUCTS FROM E COLI

Recombinant protein production from E coli started in the

late 1970s and early 1980s with insulin as the first

recom-binant protein product (9) In addition to recomrecom-binant

proteins for biopharmaceutical use, E coli is also used to

produce other recombinant proteins such as enzymes for

technical applications, polysaccharides and other

biopoly-mers such as plasmid DNA, amino acids, and primary

me-tabolites such as organic acids The production principles

and the overall procedures are similar to the recombinant

protein production procedure as described in section 10.2

The differences are in the medium composition, the growth

strategy, and production variables such as pH, DO

concen-tration, temperature, cell density, and length of cultivation

Below are some examples of processes for production of

biological products other than recombinant proteins One

is the production of the amino acid l-alanine by genetically

engineered E coli, the second is the conversion of ferulic

acid to vanillin by recombinant E coli, and the third is

the production of polysialic acid by a selected E coli K1

strain Two more examples are associated with two new

products: one is plasmid DNA and the other is a precursor

of artemisinin, a promising antimalarial drug, which is

be-ing produced in a two-phase partitionbe-ing bioreactor, a novel

approach for the biosynthesis of organic compounds

• Production of the amino acid L -alanine by metabolically

engineered E coli (39) The modified E coli strain is

grown anaerobically on a defined medium of the following

composition (14), in mmol liter1: 19.92 (NH)HPO,

7.56 NH4H2PO4, 2.0 KCl, 1.5 MgSO47H2O, and 1.0 betaineKCl The following concentrations are in μmol liter1: 8.8 FeCl36H2O, 1.26 CoCl26H2O, 0.88 CuCl2H2O, 2.2 ZnCl2, 1.24 Na2MoO42H2O, 1.21

H3BO3, and 2.5 MnCl24H2O The trace metal stock so-lution (1,000 times concentrated) is prepared in 120 mmol liter1 HCl The concentration of the carbon source glucose is 120 g liter1 The growth is carried out at 37°C and the pH is maintained at 7 by automatic addition of 5 mol liter1 NH4OH The product accumulates in the me-dium The major differences between this process and the

recombinant P aeruginosa exotoxin A production process

described above are the utilization of defined medium, the anaerobic growth, and the high concentration of glucose

• Production of vanillin Vanillin, an organic compound

with the formula C8H8O3, is a flavoring agent used in foods and beverages The compound is produced by

genetically engineered E coli by conversion of ferulic

acid (2) The process has two phases: in the first phase,

the engineered E coli strain is grown in a complex

medium to generate the required biomass, and in the second step, the cells are collected and resuspended in

a buffer containing ferulic acid The suggested process is

as follows (i) The cells are grown in LB medium (10 g liter1 tryptone, 5 g liter1 yeast extract, and 5 g liter1 NaCl), containing 25 mg liter1 tetracycline, at 37°C

in an aerated bioreactor (ii) At the end of this growth phase, the cells are collected, washed, and resuspended

at a concentration of 4 g liter1 in M9 saline/phosphate buffer (4.2 mmol liter1 Na2HPO4, 2.2 mmol liter1

KH2PO4, 0.9 mmol liter1 NaCl, and 1.9 mmol liter1

NH4Cl) containing 0.5 mg liter1 yeast extract and

5 mmol liter1 ferulic acid (iii) The cells, now in a rest-ing phase, convert the ferulic acid to vanillin This way, 2.5 g liter1 vanillin can be produced (2)

• Production of long-chain polysialic acid Polysialic acid is

a polymer that is being investigated as a potential addi-tive to different biomedical applications such as tissue engineering In the following example, the production

process of this polymer from E coli is described (24) In this process, the selected E coli K1 strain is grown in a

de-fined medium containing 1.2 g liter1 NaCl, 1.1 g liter1

K2SO4, 13 mg liter1 CaCl2, 0.15 g liter1 MgSO4·7H2O,

1 mg liter1 FeSO4·7H2O, 1 mg liter1 CuSO4·5H2O, 6.67 g liter1 K2HPO4, and 0.25 g liter1 KH2PO4 Additionally, the medium includes 13.3 g liter1 glucose and 10 g liter1 (NH4)2SO4 The cells are grown aerobi-cally (the DO is measured throughout the cultivation) at

pH 7.5 and 37°C The overall process lasts 25 h, the bac-teria grow exponentially for 10 h, and the polysialic acid accumulates during the following 15 h Following produc-tion, the cells are removed by continuous centrifugation and the polysialic acid is recovered from the supernatant fluid

• Production of plasmid DNA Plasmid DNA is produced

by E coli in a process similar to recombinant protein

production The plasmid DNA is amplified when the culture temperature is increased to 42°C The cells are initially grown at 30°C to high cell density After the completion of this growth phase, plasmid DNA produc-tion is commenced by increasing the temperature to 42°C for several more hours (20, 38)

• Production of amorpha-4,11-diene This compound is a

precursor of the new antimalarial drug artemisinin (6)

This natural product is made in E coli containing a

heterologous nine-gene pathway (18) The production

strategy, and production variables such as pH, DO

concen-tration, temperature, cell density, and length of cultivation

Below are some examples of processes for production of

biological. .. liter1 On the other hand, magnesium phosphate has a very low solubility, and therefore for high-cell-density cultivations it is recommended to add the majority of

TABLE Defined... rate (Mset) of 0.12 h1 is chosen, formation

of acetic acid should not be observed For temperature-induced production of recombinant proteins, the desired specific