DEPARTMENT : BIOTECHNOLOGY

NAME : HLATYWAYO FELICITY

REG NUMBER : H210657V

COURSE NAME : BIOPROCESS ENGINEERING

COURSE CODE : SBT 2102

ASSIGNMENT : ONE

LECTURER : DR W. MAVENGERE
1. According to the reaction, production of 1gmol of glutamic acid requires 1,5 gmol
oxygen. Therefore, producing 0,102 gmol glutamic acid requires
𝑚𝑎𝑠𝑠
moles = 𝑚𝑟

15
=147

=0.1020240 moles of glutamic acid

1 mole of glutamic acid = 0.1020240 moles

3/2 moles of oxygen =? (more)
3
2
×0.1020240

=0.153061224 moles of oxygen

Mass =moles ×mr

= 0.153061224×32
= 4.897959184g of oxygen
= 4.9g

2. SUMMARY ON THE BIOPROCESS OPTIMISATION FOR HIGH CELL DENSITY ENDOINULINASE

PRODUCTION FROM RECOMBINANT ASPERGILLUS NIGER.

RELEVANCE OF THE STUDY

An essential polyfructose hydrolyzing enzyme called endoinulinases is used to make
fructooligosaccharides from inulin. The utilization of A. niger in the synthesis of endoinulinases
makes this work relevant. Rapid growth, strong production and secretion capacities, as well as
the capability for post translational modifications, are all characteristics of Aspergillus sp.

AIMS AND OBJECTIVES

The purpose of this study was to create and evaluate a recombinant A. niger strain's
capacity to produce endoinulinases using a glucose-limited fed batch exponential
fermentation method and to learn more about the variables that affect and the difficulties
associated with producing recombinant endoinulinases from A. niger during high cell
density fermentation. On biomass growth and enzyme synthesis in fed-batch culture, the
impacts of the bioprocess parameters, glucose feed concentration, nitrogen sources
concentration, and growth rate, were examined.

METHODOLOGY
Analytical grade chemicals were used throughout. All DNA alterations and E. coli transfor-
mations were carried out in accordance with standard procedures. SDS-polyacrylamide gel was

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used to separate the proteins in the supernatant samples. The strains' stock cultures were kept in
30% (v/v) glycerol as a cryoprotectant and kept at -80 C. Spore plates were used to produce A.
niger spores. In 14 L Bio Flo 110 bioreactors, batch cultures of enzymes were produced. In order
to assess the impacts of growth rate on biomass and enzyme concentrations, an exponential feed-
ing approach was used. Fermentation in fed batches was used to measure the activity of en-
doinulinases. Fed batch fermentations (it refers to a method of feeding fresh medium or certain
nutrients intermittently or continuously during the process of batch fermentation) were carried
out at a constant exponential growth rate for the glucose feed concentration. A broth culture of
the A. niger host strain that had not undergone transformation with inulinase genes was used as
the negative control.

RESULTS
According to the molecular weight marker, endoinulinases with a molecular weight of
around 66 Kad (28) were found. As a result, the extracellular endonucleases were secreted
by the recombinant A. Niger and overexpressed. When the concentration of the glucose feed
was increased, both the biomass concentration and the volumetric activity of the enzyme
practically doubled. The recombinant A. Niger strain's morphology remained in pellet form
throughout the culture process, and this was consistent with DO levels that were kept in the
culture broth during fed batch fermentation at around 30% of saturation. In contrast, the
fungal morphology during fed batch fermentation with a concentrated feed drastically
changed from pellet to mycelia form. However, throughout the exponential development
phase, the DO levels and agitation altered.

DISCUSSION AND CONCLUSION

In order to minimize the viscosity restrictions connected with fungal growth in mycelial, the
culture conditions can be modified to stimulate fungal growth in pellet form. Due to fast oxygen
consumption, high cell densities cause both increased broth viscosity and low DO levels in the
culture. Because of the former, there is insufficient mixing, oxygen and nutrient diffusion, and
pellet disintegration as a result of internal resistance. Due to the impact on electrostatic forces that
support the integrity of the pellet, it has been theorized that changes in culture conditions linked to
insufficient mixing, such as pH, cause pellet disintegration. At high cell densities, broth viscosity
also raises the likelihood of collision and friction between the pellets, which in turn reduces the
strength of the hydrophobic and electrostatic contacts that maintain the pellet structure. However,
with high biomass concentrations, high agitation rates result in shear forces that lead to pellet
breakdown and expansion in mycelial, which further alter broth viscosity. Increasing agitation is
important to promote mixing efficiency, oxygen and nutrient diffusion. Recombinant
endoinulinases are created from A. Only at rapid growth rates was the niger strictly connected with
growth. High maintenance requirements may have contributed to poor specific growth rates by
reducing the amount of enzyme produced. Low DO levels during fed-batch feeding caused by high
biomass concentrations prompted a switch from pellet to hyphae morphology. The growth-
associated enzyme was still being made by A. niger, which continued to expand and required no
upkeep. Furthermore, at this crucial level, which was characterized by excessive viscosity and
pellet disintegration, enzyme synthesis persisted with no changes in productivity.

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REFERENCES

1. Cheng, L., Wu, J., Hen, T. (2002) A pseudo-exponential feeding method for control
of speci c growth rate in fed-batch cultures. Biochem. Eng. J. 10, 227-232.

2. Rose, S. H., van Zyl, W. H. (2008). Exploitation of Aspergillus niger for the
Heterologous production of cellulases and hemicellulases. Open. Biotechnol. J.
2, 167-175.

3. Johansson, E., Prade, T., Angelidaki, I., Svensson, S., Newson, W. R., Gunnarsson, I.
B., Hovmalm, H. P. (2015) Economically viable components from Jerusalem artichoke
(Helianthus tuberosus L.) in a biorenery concept. Int. J. Mol. Sci. 16, 8997-9016.