PART 1 - Design and Analysis of Bioreactors

Question 1

The __________________ differs from the traditional batch process in that nutrients are added in stages to maximize cell growth.

Answer 1

fed-batch process

Question 2

Features of a Fed-Batch Cultivation

Answer 2

1. A fed-batch culture is a semi-batch operation
2. Nutrients are fed either intermittently or continuously during the course of otherwise batch operation
3. The culture broth is harvested only at the end of the operational period (full
   volume), either fully or partially
4. Repeated fed-batch till the cells remain fully viable and productive
5. One or more feed streams but no effluent
6. Manipulating the feed rates during the run
7. The culture volume increases continuously
8. A dynamic operation
9. Concentrations of limiting nutrients in the culture can be manipulated
10. Nutrient concentration profile: can remain at a constant level to follow a
    predetermined profile
11. A batch mode is used to end the fermentation (maximum product titer and
    yield)
12. Manipulation of one or more feed rates, a means of regulating the nutrient
    concentration controlling the key reaction rates. What compounds should be
    fed and how they should be added

Question 3

Simple Case of Fed-Batch

Answer 3

1. Maximize the cell formation rate for constant cell mass yield
2. The substrate concentration maintained at a value that maximizes the specific the growth rate
3. Maximum cell concentration achieved at the end of the run
4. Feed rate regulated to hold the substrate concentration constant at the desired value until the reactor is full
5. Once the fermenter is full, it is run in a batch mode.

Question 4

Advantages of Fed-Batch Reactor

Answer 4

1. Intermittent feeding of substrate and maintaining low substrate concentration
   to prevent substrate inhibition
2. High Cell density
3. Catabolite repression (penicillin fermentation)
4. Crabtree effect (baker’s yeast fermentation)
5. Auxotrophic mutants, expression control of genes (inducers and repressors)
6. Extension of operation time, supplement of water lost by evaporation and
   decreasing viscosity of culture broth.

Question 5

The analysis of fed-batch with substrate continuously fed and no output: At
t=0,

Answer 5

F is constant

Question 6

The ________________, observed in certain microorganisms like baker’s yeast (Saccharomyces cerevisiae), refers to the phenomenon where these organisms preferentially ferment sugars to ethanol even in the presence of oxygen.

Answer 6

Crabtree effect

Question 7

Catabolite repression is advantageous in fed-batch reactors when the emphasis is on _____________________________________ to achieve specific bioprocess goals, such as high biomass production or the synthesis of a particular product.

Answer 7

controlled and efficient utilization of preferred carbon sources

Question 8

____________________ is a regulatory mechanism in microorganisms (such as bacteria and yeast) that involves the inhibition or repression of the expression of certain genes involved in the metabolism of alternative carbon sources when a preferred carbon source is available.

Answer 8

Catabolite repression

Question 9

__________________ is only possible if the enzymes can be easily retrieved from the product stream exiting the reactor.

Answer 9

Enzyme recycling

Question 10

Regardless of which technique is used, the common goal is to keep the appropriate __________________ within the CSTR.

Answer 10

enzyme concentration

Question 10

(CSTF)

Answer 10

continuous stirred-tank fermenter

Question 10

The cell concentration in a single chemostat can be increased by ________________ in the product stream back to the reactor.

Answer 10

recycling the biomass

Question 11

______________________ is characterized by containing a homogeneous liquid phase into which nutrients are continuously fed and from which the suspension of microorganisms and depleted feed are continuously removed.

Answer 11

The continuous stirred-tank fermenter (CSTF)

Question 12

Because the fermenter is well mixed, samples taken from any location in the fermenter will be ________ and the composition of the exit stream will be identical to that of the liquid in the fermenter.

Answer 12

identical

Question 13

In a chemostat, the flow rate is set at a ___________ and the rate of growth of the culture adjusts to this flow rate.

Answer 13

fixed value

Question 13

Continuous Stirred-Tank Fermenter (CSTF) have many applications, a few of which are listed below.

Answer 13

Pharmaceuticals: Used to study a number of different bacteria, a specific
example being analyzing how bacteria respond to different antibiotics.
Bacteria are also used in the production of therapeutic proteins such as insulin
for diabetics.
2. Manufacturing: Used to produce ethanol, the fermentation of sugar by bacteria
takes place in a series of chemostats. Also, many different antibiotics are
produced in chemostats.
3. Food Industry: Used in the production of fermented foods such as cheese.
4. Research: Used to collect data to be used in the creation of a mathematical
model of growth for specific cells or organisms.

Question 13

As the concentrations in the chemostat adjust to the feed rate, the _____________ remains constant, and a steady-state is established.

Answer 13

dilution rate

Question 14

In a turbidostat, the ______________ is kept constant by setting the outlet flow rate equal to the inlet flow rate; however, the inlet flow rate is modified to maintain a constant biomass concentration.

Answer 14

liquid volume

Question 15

As a result, the dilution rate in a turbidostat adjusts to the ________________ needed to produce the required biomass concentration.

Answer 15

steady-state value

Question 15

It is easier to operate chemostat than turbidostat, because the former can be
done by ____________________, whereas the latter requires an _________________________.

Answer 15

setting the pump at a constant flow rate

optical sensing device and a controller

Question 16

However, the _____________ is recommended when continuous fermentation needs to be carried out at high dilution rates near the washout point, since it can prevent washout by regulating the flow rate in case the cell loss through the output stream exceeds the cell growth in the fermenter.

Answer 16

turbidostat

Question 17

The chemostat setup consists of a

Answer 17

sterile fresh nutrient reservoir connected to a growth chamber or reactor.

Question 17

Fresh medium containing nutrients essential for cell
growth is pumped continuously to the chamber from the medium reservoir. The
medium contains a specific concentration of ______________________, which allows for a maximum concentration of cells within the growth chamber.

(CHEMOSTAT)

Answer 17

growth-limiting nutrient (Cs)

Question 18

Temperature and pressure must also be controlled within the chemostat in order to maintain optimum conditions for cell growth. Using a ___________ for the growth chamber allows for easy temperature control.

Answer 18

jacketed CSTR

Question 18

Some processes such as biological fermentation are quite exothermic, so _________________ is used to keep the temperature at its optimum level. As for the reactor pressure, it is controlled by an exit air stream that allows for the removal of excess gas.

Answer 18

cooling water

Question 18

Another means of controlling the steady state cell concentration is _______________________ at which the medium flows into the growth chamber.

The medium drips into culture through the air break to prevent bacteria from traveling upstream and contaminating the sterile medium reservoir.

Answer 18

manipulating the rate

Question 18

Varying the concentration of this growth-limiting nutrient will, in turn, change the _____________________________

Answer 18

steady state concentration of cells (Cc).

Question 19

For aerobic cultures, purified air is bubbled throughout the vessel’s contents by a _________. This ensures enough oxygen can dissolve into the reaction medium. For anaerobic processes, there generally is not a need for an air inlet, but there must be a _____________ in order to prevent a build-up in pressure within the reactor.

Answer 19

sparger

gas outlet

Question 20

In order to prevent the reaction mixture from becoming too acidic (cell respiration causes the medium to become acidic) or too basic, which could hinder cell growth, a _______________ is needed in order to bring pH balance to the system.

Answer 20

pH controller

Question 21

The stirrer ensures that the contents of the vessel are well mixed. If the stirring speed is too high, it could damage the cells in culture, but if it is too low, gradients could _________ in the system.

Significant gradients of any kind (temperature, pH, concentration, etc.) can be a ______________ to cell production and can prevent the reactor from reaching steady state operation.

Answer 21

build up
detriment

Question 22

Another concern in reactor design is fouling. Fouling is generally defined as the
deposition and accumulation of unwanted materials on submerged surfaces or surfaces in contact with fluid flow. When the deposited material is biological in
nature, it is called _______________.

Answer 22

biofouling

Question 23

To minimize these effects,
industrial chemostat reactors are commonly ________________, containing volumes of up to
1300 cubic meters, and are often constructed from stainless steel. The ________________________ allow for easy cleaning.

FOULING

Answer 23

cylindrical

cylindrical shape and smooth stainless-steel surface

Question 24

Control Factors of a Chemostat

Answer 24

1. pH level
2. Temperature
3. Dilution Rate
4. OTR
5. Agitation Speed

Question 24

The operators need to determine an optimal pH and maintain the CSTR at it for efficient operation.

Controlling the pH at a desired value during the process is extremely important because there
is a tendency towards a lower pH associated with cell growth due to cell
respiration (carbon dioxide is produced when cells respire and it forms ___________________ which in turn causes a lower pH).

Under extreme pH conditions, cells ________________, therefore appropriate action needs to be taken to restore the
original pH (i.e. adding acid or base).

Answer 24

carbonic acid
cannot grow properly

Question 24

When the dilution rate is greater than the specific growth rate (D > μ), the
dCC/dt term becomes

Answer 24

negative.

Question 25

One of the important features of the chemostat is that it allows the operator to ________________________. The most common way is controlling the _____________, although other methods such as controlling temperature, pH or oxygen
transfer rate can be used.

Answer 25

control the cell growth rate

dilution rate

Question 25

While using a chemostat, it is useful to keep in mind that the specific
growth rate of bacteria _______ the dilution rate at steady state.

Answer 25

equals

Question 26

This shows that the concentration of cells in the reactor will decrease and
eventually become zero. This is called ___________, where cells can no longer maintain themselves in the reactor.

Answer 26

wash-out

Question 27

The chemostat is a very
convenient tool to study the growth of specific cells because it allows the
operators to control the _____________________________.

Answer 27

amount of oxygen supplied to the reactor

Question 27

A __________, usually automated and powered with a motor, mixes the contents of the
chemostat to provide a homogeneous suspension.

Answer 27

stirrer

Question 27

The specific production rate of biomass by the CSTF is the product of the biomass
concentration and the volumetric flow rate of feed divided by the volume of the
fermenter.

Answer 27

PRODUCTIVITY OF CHEMOSTAT

Productivity = DX

Question 28

By reducing the effects of
process disruption, cell recycling improves ____________________ (or stability) in some systems (such as waste-water treatment).

Answer 28

productivity and the rate of conversion

Question 29

“a chemostat can be operated at dilution rates __________ than the specific growth rate when cell recycle is used.”

Answer 29

higher

Question 30

Methods of Cell Recycling in Fermentation Processes

Answer 30

1. External Biomass Feedback
2. Perfusion Culture or Internal Biomass Feedback

Question 31

In this method, a cell separator such as a centrifuge or settling tank is
used to concentrate the biomass leaving the reactor.

Answer 31

External Biomass Feedback

Question 32

A portion of the
______________is recycled back to the CSTR with flow rate 𝐹𝑟 and cell concentration 𝑥𝑟. Such systems can be operated under steady-state conditions and are used extensively in biological waste treatment.

Answer 32

concentrate

Question 32

This setup of cell recycling is often used for mammalian cell culture since there is a depletion of nutrients and accumulation of inhibitory products that limit batch cell densities for many animal cell lines of about 10 cells ml−1.

Answer 32

Perfusion Culture or Internal Biomass Feedback

Question 33

The use of CSTR with recycling encompasses two effects:

Answer 33

1. The substrate concentration in the effluent will increase.
2. Operating this system in practice may tend to be unstable.

Question 33

One solution to this problem is to devise a method to retain the biomass in the
fermenter while allowing the spent feed to pass out. Several methods of achieving this are as follows:

Answer 33

1. Use of Centrifuge
2. Use of Internal Filter
3. Use of an External Cross Flow Filter

Question 34

In wastewater treatment systems, it is typically desirable to operate in a
continuous fermenter at ___________________

Answer 34

high dilution rates (↑ 𝐷).

Question 35

With growing cells, if all the biomass is recycled or retained in the reactor,
the cell concentration will increase with time and _____________ will not be achieved. Therefore, for steady-state operation, some proportion of the biomass must be ___________ from the system.

Answer 35

steady state
removed

Question 36

The operation of the system outlined in the process layout is analyzed by taking material balances over the fermenter vessel.

It is assumed that in this idealized case, there is _________________________ occurring in the separator, so that the substrate concentration 𝑆 in the entering stream is the same as that in the clarified liquid effluent stream, recycle stream, and exit biomass rich stream.

Answer 36

no biochemical reaction or growth

Question 37

When multiple CSTRs are connected in series, it creates a multi-stage process where conditions, such as pH, temperature, and limiting nutrients, in each reactor can be controlled _________________.

Answer 37

independently

Question 37

CSTR CALCULATIONS STEP-BY-STEP

Answer 37

1. Find Dilution Rate
2. Find Substrate Concentration
3. Find Biomass Concentration
4. Ratio of the Critical Dilution Rate to the Maximum Specific Growth Rate
5. Find Net Specific Growth Rate
6. Find Concentrations of Substrate and Biomass

Question 38

In contrast, the feed to the first reactor will have a biomass concentration of

Answer 38

zero

Question 38

Second reactor will not receive a

Answer 38

sterile feed

instead, it will contain biomass and substrate at concentration of Xi and Si