3-weeks course 27032: Microbiology and Fermentation Technology

Simulation Exercise: General information, outline and task-list

Simulation Exercise: General information, outline and task-list....................................................... 1

General instructions .................................................................................................................... 3

Guidelines for report writing ....................................................................................................... 3

Task 1. Getting started................................................................................................................. 4

Task 2. Substrate inhibition kinetics. ........................................................................................... 5

Task 3. Antibiotic Production...................................................................................................... 6

Task 4. Designing a chemostat operation..................................................................................... 8

Task 5. Fed-batch process by using yeast. ................................................................................. 10

Task 5-A. Chemostat process for protein production ............................................................. 10

Task 5-B. Fed-batch process for baker’s yeast production ..................................................... 10

Task 6. Mystery of a "continuous stirred overflow tank reactor"................................................ 12

Availability of assistance

Teaching assistance will be provided from Monday to Thursday from 9:00 to 12:00. You are
welcome to contact Kiran, Louise or Ana Rita between 13:00-17:00, either by email or by an
appointment (fix by phone/email).

Kiran Raosaheb Patil

Email: krp@biocentrum.dtu.dk

Phone: 4525 2703/2677 (O), 26204296 (M)

Office: Room 226/126, Building 223.

Louise Mølgaard

Email: lom@biocentrum.dtu.dk

Phone: 45252699(O)

Office: Room 218, Building 223.

Ana Rita Brochado

Email: arb@biocentrum.dtu.dk

Phone: 45252997(O)

Office: Room 213, Building 223.

General instructions

1. You can work either in groups or individually. However, the report must be handed in on
individual basis.

2. Monday 16th June, before you start your experiments, will be the deadline for receiving
reports. Hand them to your teacher in-charge of the experiments.

3. Note that you will use some of the results from the simulation exercise for you laboratory
work with yeast fermentations.

Guidelines for report writing

1. Please do not write a very long report. An ideal report should not be more than 15 A4 pages.

2. Use tables, figures etc. instead of long descriptions to increase the clarity and to save space.

3. Whenever necessary, write down the appropriate equations used in the simulations.

4. Provide justifications for the choices made. For example, if you suggest that a continuous
fermentation is better than batch fermentation, provide the reasons you used to arrive at this
recommendation.

5. It is not necessary, and is discouraged, to submit the MATLAB programs (either provided to
you or written/modified by you) with the report.

6. Make a separate sub-section in your report for each task.

7. Submit reports in print, please do not email them.

Task 1. Getting started.

Briefly list different industrial/natural processes/products where design of bio-reactors and

optimization of their operation is of relevance (Minimum 3 and maximum 5 processes). Take the
following points into consideration:

i) Is the product a high-value or low-value product? Depending on this, the design objective may be
maximization of yield, conversion of substrate or volumetric productivity of the product.

ii) In what mode (batch, fed-batch, continuous) is the process operated? Why is this mode preferred
for this process?
Task 2. Substrate inhibition kinetics.

Plot a curve showing relationship between substrate concentration ‘s’ and growth rate ‘µ’ for a)
monod kinetics; and b) substrate inhibition kinetics; both described in the following equations.
Discuss the difference between the natures of these two curves. What is the possible implication of
this difference on the design of a bio-process?

µ max ⋅ S
µ= Simple Monod growth kinetics
(S + Ks )

µ max ⋅ S
µ= 




Monod kinetics with substrate inhibition

 

S2
S + Ks +
 

 

 

Ki



Task 3. Antibiotic Production

Since Alexander Fleming discovered penicillin in 1928

this compound has revolutionized the treatment of
infectious disease.
A pharmaceutical company wants to produce penicillin
from the fungus Penicillium chrysogenum. The market
demand is a fermentor capable of producing 10.000.000
kg/year.
In order to access the productivity of the strain prior to large scale production a small scale batch
fermentation was run and the concentration of the product was measured by taking samples from
the fermentation. These were the initial small scale results.

time (h) Product (g/L) Biomass (g/L) Glucose (g/L)

0 0,0 0,1 50,0
5 0,0 0,2 49,9
10 0,0 0,4 49,8
15 0,0 0,8 49,6
20 0,0 1,6 49,2
24 0,0 2,9 48,6
28 0,0 5,0 47,5
35 0,0 13,4 43,3
40 0,0 27,0 36,5
50 0,1 27,1 36,2
60 0,2 27,2 34,7
80 0,5 27,5 25,7
100 0,8 27,6 16,7
120 1,1 27,8 7,7
130 1,2 27,9 3,2

A. What is the volumetric productivity (qp)? Calculate both the overall volumetric productivity
and the volumetric productivity in the “production phase”.

B. Discuss what you should take into consideration when up-scaling a fermentation process
from lab-scale to production scale?

Assume that you can run a batch fermentation in one week, where the bioreactor is running 6
days in total with 2 days of biomass formation, 4 days of maximum productivity and one day
doing cleaning. Furthermore due to holidays the fermentation plant is shut down 3 weeks a year.
 C. How big a fermentor would you need to meet market demands?
(Assume that the usual size of a “large” fermentor is 500 m3)

The cost of our substrate is 100 DKK/kg and Ysp=0,03 g penicillin / g substrate. The running cost of
running the fermentation is 10 DKK/L/h.

D. Calculate what price penicillin (per gram) should be sold at to break even with the running
costs?
E. If the pharmaceutical company wants to have a profit of at least 86% what would the price
of the product be?
F. Discuss different methods that could be used to increase profit?
Task 4. Designing a chemostat operation.

Brian is a biological engineer and he works in a company for biomass production. A certain day, his
boss comes to talk to him and gives him a new task: to produce a valuable microbial biomass (used
as a starter culture for dairy products) using a chemostat mode of operation. To start, Brian’s boss
supplies him some information about the fermentation conditions and what fermentor to use. It is a
10 L fermentor, the substrate concentration in the feed is 10 g/l and the yield of biomass growing in
the given substrate is 0.5 g DW/ g subst. The mixing conditions and the growing temperature of the
microorganism are already known, so Brian is ready to start. However, when he is in the plant to get
everything ready and start the fermentation, he realizes that his boss totally forgot to give him one
parameter… what is the flow rate he should use? Then, Brian decides to go and ask his boss this
small number and he will know it by heart, as everything else! However, the boss says: “That is
YOUR task!”

a) After thinking for a while, he concludes that the best way to find the flow rate to use is to go to
the plant and try different flow rates. At the same time, he collects some data. S is the substrate
concentration inside the fermentor, F is the feed rate and X is the biomass concentration inside the
fermentor.

F (l/h) S (g/l) X (g DW/l)

0.05 0.01 4.99
0.5 0.13 4.94
2 0.83 4.59
2.5 1.30 4.35
3 2.13 3.94
3.5 3.87 3.07
4.2 10 0

His first thought is that a very low flow rate should be used. However, after some reflection he
decides to check how much biomass in g DW he has in one hour for each flow rate he tested. What
do you think he will conclude?

b) Brian decides to study this situation more in detail. So, he guesses that this situation might be
related with growth kinetics. Two different microbial growth models that may be valid in this
situation are Monod and Contois. Determine the kinetic parameters, Ks and µ max, and wash-out
dilution rate for each model. What is the yield YXS? Does any of the two models seems to be more
appropriate? Start by writing the mass balances.

Cs
µ = µ max
Cs + K s

Cs
µ = µ max
Cs + K s ⋅ X

c) Comment on differences between the kinetic models Contois and Monod, as well as their
application.
Task 5. Fed-batch process by using yeast.

The yeast Saccharomyces cerevisiae is widely used in different industrial applications ranging from
baking, brewing to therapeutic protein production. A fed-batch production process is indispensable
in applications where either high yield/productivity of biomass/proteins is desirable. One of the
major reasons for this choice is so called crab-tree effect or overflow metabolism. In a simplified
model the overflow metabolism can be seen as diversion of excess glucose-uptake to fermentative
pathway (ethanol production) when yeast is exposed to high glucose levels. Thus, biomass yield
drops dramatically at high glucose uptake rates due to ethanol production. This is of course
undesirable when the primary objective is to produce biomass. A solution to this problem is to use
fed-batch or chemostat mode of operation.

Task 5-A. Chemostat process for protein production

The article by Carlsen et al. (Biotechnology & Bioengineering, 1996) presents a study on protein
production by using chemostat operation. It uses a simple model for simulating over-flow
metabolism.

1. Briefly describe the kinetic model used, especially in relation to overflow metabolism. What is
Dcrit in this model, i.e. dilution rate at which ethanol formation is onset in an aerobic chemostat?
What is Dmax? Verify your results by using figure 3.

2. Discuss why the optimal dilution rate for protein production equals the critical dilution rate
(figure 6). What dilution rate you would recommend to use in a production process?

Task 5-B. Fed-batch process for baker’s yeast production

It is desired to produce baker’s yeast using a fed-batch. The kinetics and mass balances for batch &
fed-batch process are provided to you as MATLAB files. Use “Batch_saccharomyces.m” and
“Fedbatch_saccharomyces.m” for simulating Batch and Fed-batch respectively. You can change
certain conditions and parameters in the file. Please ask teaching assistants for help using these files
for simulations. Refer to the article by Villadsen & Patil (Biotechnology & Bioengineering, 2007)
for fed-batch equations. Note that you will run batch and fed-batch fermentations with yeast in this
course. You should use these simulations to decide what should be the feeding profiles to obtain
maximum biomass productivity (gDW/hr/L) in a fed-batch process.
1. First simulate a batch process with 10 g/L of initial glucose and 0.01 gDW/L of inoculum (initial
biomass). Calculate yield of biomass and ethanol on glucose. How does the biomass yield compare
with that in a chemostat process run below critical dilution rate? Comment on the difference.

2. Use the glucose, ethanol & biomass concentration at the end of batch phase to simulate fed-batch.
Devise a feeding strategy (such as constant feed, linear feed, exponential feed, or combination of
these) so as to obtain maximum biomass productivity. The feed glucose concentration is 100 g/L,
while the initial volume (batch phase) is 3.5 L and the final volume will be 4.5 L. Consider two
scenarios.

A) Assume that O2 transfer is not a limitation. Calculate the maximum value of Kla required in
the process that you suggest.

B) Now, assume that the maximum Kla value that can be achieved for the given reactor is 600
hr-1; and that the dissolved oxygen concentration needs to be at least 60% of the saturation
value.

Plot glucose concentration, volume, biomass, ethanol, Kla and growth rate as function of time in all
simulations that you try (report results for at least 5 different feeding schemes). Justify your choice
of best feeding strategy in both cases.

3. Which situation (A or B) is industrially more relevant? Why?

4. Is fed-batch the best option in this case? Justify your answer.

Task 6. Mystery of a "continuous stirred overflow tank reactor".
(Borrowed from “Chemical Reaction Engineering” by Octave Levenspiel)

Holmes: You say he was last seen tending1 this vat2 ....
Sir Boss: You mean "continuous stirred overflow tank reactor" Mr. Holmes.
Holmes: You must excuse my ignorance of your particular technical jargon, Sir Boss.
Sir Boss: That's all right. However, you must find him, Mr. Holmes. Imhibit was a queer3 chap4;
always staring into the reactor, taking deep breaths and licking his lips, but he was our very best
operator. Why, since he left, our conversion of googliox has dropped from 80% to 75%.
Holmes (tapping the side of the vat idly): By the way, what goes into the vat?
Sir Boss: Just an elementary reaction between ethanol and googliox, if you know what I mean.
Of course, we maintain a large excess of alcohol, about 100 to 1 and ....
Holmes (interrupting): Intriguing, we checked every possible lead in town and found not a single
clue.
Sir Boss: We'll give the old chap a raise - about twopence per week - if only he'll come back.
Watson: Pardon me, but may I ask a question?
Holmes: Why certainly, Watson.
Watson: What is the capacity of this vat, Sir Boss?
Sir Boss: A hundred Imperial gallons5, and we always keep it filled to the brim. That is why
we call it an overflow reactor. You see we are running at full capacity - profitable
operation you know.
Holmes: Well, my dear Watson, we must admit that we're stumped6, for without clues
deductive powers are of no avail.
Watson: Ahh, but that is where you are wrong, Holmes. (Turning to the manager) Imhibit was
a largish fellow - say about 18 stone7- was he not?
Sir Boss: Why yes, how did you know?
Holmes (with awe): Amazing, my dear Watson!
Watson (modestly): Why it's quite elementary, Holmes. We have all the clues necessary to deduce
what happened to the happy fellow.

With Sherlock Holmes and Sir Boss impatiently waiting, Dr. Watson casually leaned against the
vat, slowly and carefully filled his pipe and - with the keen sense of the dramatic - lit it. There our
story ends.

(a) What happened to Imhibit?

(b) How did Watson estimate his weight? What assumption did he make to arrive at that
value?

1
Operating/Taking care of
2
Tank
3
Strange
4
Man
5
1 Imperial gallon = 4.546 lit
6
Failed/Clueless
7
1 Stone = 6.4 Kg