Bioreactor

Learning outcomes: Familiarize with

1. Workings of a Bioreactor
2. Aseptic technique
3. Monitoring growth of cells in a bioreactor using a spectrophotometer relating OD
measurements to growth.
4 Serial dilution
5. Monitoring growth and growth curves
Bioreactor
Bioprocessing Part 1: Fermentation – YouTube
This video describes the role of the fermentation process in the creation of
biological products and illustrates commercial-scale fermentation at a cellular level.
Included are descriptions of types of fermentation (intracellular, extracellular),
types of cells (aerobic, anaerobic), and cell nutrition

1a Based on the video- name 4 products created in commercial fermentation.

1b What is scaling up?
1c Is fermentation upstream or downstream?
1d What are the key factors to control in a fermentar?
1e Name at least 4 structures in the fermentor with screen shots of the region.
1f What is the BPR?
1g What is referred to as the broth?
 Depending on the type of cell and the product that
you want you have to control the conditions in the
fermentor.
YEAST METABOLISM
Using glucose, yeast Saccharomyces cerevisiae has three major pathways for growth.
(1) The fermentation of glucose normally takes place in the absence of oxygen or when the glucose
concentration is high. Under such condition, cells only achieve a maximum specific growth rate of ~0.45
hr–1 with a low biomass yield of 0.15 g dry mass per gram glucose consumed. The respiratory quotient
(i.e., the ratio of CO2 production rate to the O2 consumption rate) is high with a net yield of 2 ATP per
mole of glucose metabolized. C6H12O6 → 2C2H5OH + 2CO2 + 
(2) The oxidation of glucose predominates when the glucose concentration is below 50 mg/L in an
aerobic condition. A maximum specific growth rate of only ~0.25 hr–1 with a biomass yield of 0.5 g dry
mass per gram glucose consumed is attained. Under the aerobic reaction, a respiratory quotient of
about 1, and a high energy yield of 16-28 ATP per mole of glucose metabolized.
C6H12O6 + 6O2 → 6CO2 + 6H2O + 
(3) The oxidation of ethanol only occurs when fermentative substrates are not available or in very
limited supply. The cells only attain a maximum growth of ~0.20 hr–1 with a high biomass yield of
about ~0.6-0.7 g dry mass per gram ethanol consumed, and a low respiratory quotient of about 0.7. An
energy yield of ~6-11 ATP per mole of ethanol metabolized. C2H5OH + 3O2 → 2CO2 + 3H2O + 
Sterile technique is ESSENTIAL when working with microorganisms! It is important
to protect strains from contamination with other strains and from the many
undefined microbes in the environment. Large numbers of diverse
microorganisms are all around us - in the air, on laboratory surfaces, on your skin
and on your clothing. True to their name, microorganisms are too small to be
detected by the eye, but they grow rapidly in laboratory culture media. Correct
transfer techniques and the use of sterile reagents are usually enough to prevent
contamination.

2. Watch this video, and state how sterile sampling was done. Suggest
TWO(2) other steps not mentioned in the video, but maybe that you
have learnt about previously on steril technique.

https://www.youtube.com/watch?v=bVe6QdvYoj8
Measuring growth of population

- Indirect method absorbance, as more cells

If a culture is placed in a cuvette, a light beam

passing through the cuvette will be scattered more
or less by the cells, depending on the cell density;
to our eye this appears as turbidity.
The scattering intensity or turbdity, often termed
optical density (OD), is proportional (∼) to the cell
density:
OD600 measurement provides information about microbial growth
Glass or Quatz cuvette can be used for measurement

Note: The method does not discriminate between living and dead cells. The light
scattering ability of a cell depends on its size and geometry, so the same number of
yeast cells would scatter light more than the same number of bacterial cells, because
the bacterial cells are much smaller.
Determination of Cell Concentration
You must ensure that absorbance is proportional to cell concentrations. It has
been found that is yeast if the absorbance readings are greater than 0.25,
further dilution is required. If a culture approaches such a critical OD, samples
must be diluted prior to measurement by a defined dilution factor, d. The
measured value is then divided by d to get the corrected or calculated optical
density, ODcorr.

3. Beyond what OD value is dilution required for

bacteria for which the cell density is plotted against
OD in the graph shown. Why?
That is why when we sample, we do a serial dilution so that we can obtain an
absorbance value that falls within the range where there is a linear
relationship between absorbance and cell density
A serial dilution is any dilution in which the
concentration decreases by the same factor in
each successive step.
In serial dilutions, you multiply the dilution
factors for each step.
The dilution factor or the dilution is the initial
volume divided by the final volume.
DF=Vi/Vf
For example, if you add a 1 mL sample to 9 mL
of diluent to get 10 mL of solution,
DF=Vi/Vf = 1mL/10mL=1/10. This is a 1:10
dilution.

Serial dilution
https://www.youtube.com/watch?v=Zqd
U3VfQ_Tc&feature=emb_logo
4a What is the dilution factor?
4b What is the concentration of the last tube?
We follow the OD by optical measurement and plot it versus t to obtain a growth
curve; from this we can calculate μ (specific growth rate or often simply growth rate,
(unit: d-1, h-1 or min-1)).of the bacterium. For data evaluation, we can display the
exponential curve as a straight line if we plot the OD on a logarithmic scale versus time
on a linear scale. The steepness, or gradient, of the line in a semi-logarithmic graph can
be used to determine how fast the organism is dividing, i.e. to find the doubling time,
5a Follow the instructions in the Absorbance at 660nm
following video to determine your Time Culture 1 Culture 2
(mins)
doubling time using EXCEL. Send me
0 0.05 0.05
your EXCEL file.
30 0.05 0.05
https://www.youtube.com/watch?v=f5gDkP 60 0.06 0.05
YBnRU 90 0.08 0.07
120 0.12 0.1
150 0.24 0.21
180 0.45 0.4
210 0.86 0.7
240 0.99 1.4
270 1.05 1.4
300 1.15 1.42
330 1.2 1.46
360 1.25 1.48
For a given species the doubling time, td, is a kind of species-specific,
unchangeable characteristic: every species has a particular, genetically fixed
doubling time under optimal growth conditions. The doubling time of a given b
species growing optimally can thus be regarded as a fixed value. Hence, we can
define µ: the specific growth rate or often simply growth rate, (unit: d-1, h-1
or min-1).

5b From the doubling time

you obtained earlier,
determine the specific
growth rate