Microbiology
Aim:
• practical aspects of fermentation à application of the theory in the Biochemistry section
• examples of successful microbial biotechnology à show the success of first generation microbial
biotechnology
• potential of recombinant biotechnology for the development of second and third generation
microbial biotechnology
1. Short overview of the central metabolic pathways with the focus on energy generation for
production of biomass, as well as product formation via fermentation.
2. Discussion of yeast-based fermentation processes for aerobic production of baker’s yeast and single-cell
protein and anaerobic fermentations for wine and beer making processes, as well as the production of fuel
ethanol (bioethanol).
3. Recombinant DNA technology for the production of pharmaceutical products and enzymes in microbial
hosts.
Central metabolism
- Biotechnology - is the use of biological processes to produce commodities (industrial relevant
products).
- Microbial biotechnology - is the use of microbial organisms to produce industrial relevant products.
à products produced by the organism or the microbial organism (cellular yield of biomass
production) itself.
- Metabolism - is the set of chemical reactions that occur in a cell, which enable it to live, grow and
divide.
- central metabolism - is the heart of an organism’s total metabolic capacity.
à provides the metabolic link between these pathways
à includes the Embden-Meyerhof- Parnas (EMP) pathway of glycolysis, the pentose phosphate
pathway, and the citric acid cycle, with individual variations depending on the specific organism and
the ecological niche in which the organism lives.
- Organisms use organic nutrients to :
• supply the precursors for all their components
• generate energy for biosynthesis and other processes.
à catabolic pathways - Degradative pathways where nutrients are metabolised
à anabolic pathways - biosynthetic pathways
The aerobic metabolism of glucose to C3-compounds by microorganisms can be divided in three major
steps:
AEROBIC METABOLISM OF GLUCOSE BY MICROORGANISMS (YEAST)
1. The conversion of glucose (C6-compound) to pyruvate (C3-compound).
i. Glycolysis
ii. The Embden-Meyerhof-Parnas pathway (EMPP, glycolysis)
• provides most of the energy
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, • important in the biosynthesis of for example nucleotides (ribose, deoxyribose) and thus
nucleic acids
iii. The Pentose Phosphate pathway (PPP)
• essentially for biosynthesis of pentose phosphates
• ATP and NADH + H+ (NADH2), NADPH + H+ (NADPH2) is also a very important product
à plays a key role in the biosynthesis of cellular components.
• utilization of xylose via conversion to xylulose-5-phosphate.
• cannot utilise the PPP (cannot synthesise pentoses), is overcome by synthesis of pentose
phosphates by reverse reactions of the PPP, thus xylose-5-P can be produced from 3-
phosphoglyceraldehyde and glucose-6-P.
EMPP PPP
(use more than one of these pathways to ensure optimal utilisation of glucose. The percentage usage of
these different pathways largely = depends on the environmental conditions)
2. The conversion of pyruvate to 3 CO2 molecules with the released energy stored in reduced energy-
carrying molecules.
• Released energy is stored in reduced energy-carrying molecules In presence of 02
• NADH, NADPH, FADH
• place in the Krebs cycle (tricarboxylic acid cycle or citric acid cycle (Fig. 3))
0836422883 - VN DO NOT FORWARD GENETICS215
, • The reduced energy-carrying molecules that form include ATP, NADH2 and FADH + H+
(FADH2).
• most useful source of energy is ATP.
• An additional step is necessary to convert FADH2 and NADH2 to ATP.
à takes place in the electron transport chain.
3. The generation of ATP from the reduced energy-carrying molecules in the presence of O2.
• Electron transport phosphorylation
• Electron transport chain
ELECTRON TRANSPORT CHAIN (ETC)
1. Prokaryotes = larger diversity of electron carriers than eukaryotes.
- Occurs on the plasma membrane in prokaryotes and the mitochondrial membrane in
eukaryotes.
2. end products of the ETC = H2O + H+ molecules.
3. The H+ molecules = a proton gradient outside the cytoplasmic membrane.
à This gradient promotes the ATPase reaction that leads to ATP synthesis (Chemiosmotic
hypothesis presented by Mitchell - Fig. 4B).
4. The ETC creates a proton pump that leads to ATP synthesis (and thus energy).
5. For microorganisms that contain cytochrome C (e.g. B. subtilis) 6 H+ protons are produced that
leads to the synthesis of 3 ATP molecules/NADH2 (Fig. 5A).
6. For microorganisms that contain no cytochrome C (e.g. E. coli) 4 H+ protons are produced that
leads to the synthesis of 2 ATP molecules/ NADH2 (thus, 2 H+ → 1 ATP) (Fig. 5B).
à can only generate 2 ATPs from NADH2 and 1 ATP from FADH2.
- The ETC's of microorganisms can vary considerably with regards to the cytochromes in the chain
i.e. (microorganisms that contain cytochrome C) = Pseudomonas, Gluconobacter, Acetobacter and
Micrococcus, while those without cytochrome C include Escherichia coli and Proteus (Table 2).
Table 2: Differences between the ETC of microorganisms (wavelengths absorbance spectra of the major
cytochromes are indicated).
0836422883 - VN DO NOT FORWARD GENETICS215
, Fig. 3: The tricarboxylic acid cycle (TCA or Krebs cycle)
0836422883 - VN DO NOT FORWARD GENETICS215
Aim:
• practical aspects of fermentation à application of the theory in the Biochemistry section
• examples of successful microbial biotechnology à show the success of first generation microbial
biotechnology
• potential of recombinant biotechnology for the development of second and third generation
microbial biotechnology
1. Short overview of the central metabolic pathways with the focus on energy generation for
production of biomass, as well as product formation via fermentation.
2. Discussion of yeast-based fermentation processes for aerobic production of baker’s yeast and single-cell
protein and anaerobic fermentations for wine and beer making processes, as well as the production of fuel
ethanol (bioethanol).
3. Recombinant DNA technology for the production of pharmaceutical products and enzymes in microbial
hosts.
Central metabolism
- Biotechnology - is the use of biological processes to produce commodities (industrial relevant
products).
- Microbial biotechnology - is the use of microbial organisms to produce industrial relevant products.
à products produced by the organism or the microbial organism (cellular yield of biomass
production) itself.
- Metabolism - is the set of chemical reactions that occur in a cell, which enable it to live, grow and
divide.
- central metabolism - is the heart of an organism’s total metabolic capacity.
à provides the metabolic link between these pathways
à includes the Embden-Meyerhof- Parnas (EMP) pathway of glycolysis, the pentose phosphate
pathway, and the citric acid cycle, with individual variations depending on the specific organism and
the ecological niche in which the organism lives.
- Organisms use organic nutrients to :
• supply the precursors for all their components
• generate energy for biosynthesis and other processes.
à catabolic pathways - Degradative pathways where nutrients are metabolised
à anabolic pathways - biosynthetic pathways
The aerobic metabolism of glucose to C3-compounds by microorganisms can be divided in three major
steps:
AEROBIC METABOLISM OF GLUCOSE BY MICROORGANISMS (YEAST)
1. The conversion of glucose (C6-compound) to pyruvate (C3-compound).
i. Glycolysis
ii. The Embden-Meyerhof-Parnas pathway (EMPP, glycolysis)
• provides most of the energy
0836422883 - VN DO NOT FORWARD GENETICS215
, • important in the biosynthesis of for example nucleotides (ribose, deoxyribose) and thus
nucleic acids
iii. The Pentose Phosphate pathway (PPP)
• essentially for biosynthesis of pentose phosphates
• ATP and NADH + H+ (NADH2), NADPH + H+ (NADPH2) is also a very important product
à plays a key role in the biosynthesis of cellular components.
• utilization of xylose via conversion to xylulose-5-phosphate.
• cannot utilise the PPP (cannot synthesise pentoses), is overcome by synthesis of pentose
phosphates by reverse reactions of the PPP, thus xylose-5-P can be produced from 3-
phosphoglyceraldehyde and glucose-6-P.
EMPP PPP
(use more than one of these pathways to ensure optimal utilisation of glucose. The percentage usage of
these different pathways largely = depends on the environmental conditions)
2. The conversion of pyruvate to 3 CO2 molecules with the released energy stored in reduced energy-
carrying molecules.
• Released energy is stored in reduced energy-carrying molecules In presence of 02
• NADH, NADPH, FADH
• place in the Krebs cycle (tricarboxylic acid cycle or citric acid cycle (Fig. 3))
0836422883 - VN DO NOT FORWARD GENETICS215
, • The reduced energy-carrying molecules that form include ATP, NADH2 and FADH + H+
(FADH2).
• most useful source of energy is ATP.
• An additional step is necessary to convert FADH2 and NADH2 to ATP.
à takes place in the electron transport chain.
3. The generation of ATP from the reduced energy-carrying molecules in the presence of O2.
• Electron transport phosphorylation
• Electron transport chain
ELECTRON TRANSPORT CHAIN (ETC)
1. Prokaryotes = larger diversity of electron carriers than eukaryotes.
- Occurs on the plasma membrane in prokaryotes and the mitochondrial membrane in
eukaryotes.
2. end products of the ETC = H2O + H+ molecules.
3. The H+ molecules = a proton gradient outside the cytoplasmic membrane.
à This gradient promotes the ATPase reaction that leads to ATP synthesis (Chemiosmotic
hypothesis presented by Mitchell - Fig. 4B).
4. The ETC creates a proton pump that leads to ATP synthesis (and thus energy).
5. For microorganisms that contain cytochrome C (e.g. B. subtilis) 6 H+ protons are produced that
leads to the synthesis of 3 ATP molecules/NADH2 (Fig. 5A).
6. For microorganisms that contain no cytochrome C (e.g. E. coli) 4 H+ protons are produced that
leads to the synthesis of 2 ATP molecules/ NADH2 (thus, 2 H+ → 1 ATP) (Fig. 5B).
à can only generate 2 ATPs from NADH2 and 1 ATP from FADH2.
- The ETC's of microorganisms can vary considerably with regards to the cytochromes in the chain
i.e. (microorganisms that contain cytochrome C) = Pseudomonas, Gluconobacter, Acetobacter and
Micrococcus, while those without cytochrome C include Escherichia coli and Proteus (Table 2).
Table 2: Differences between the ETC of microorganisms (wavelengths absorbance spectra of the major
cytochromes are indicated).
0836422883 - VN DO NOT FORWARD GENETICS215
, Fig. 3: The tricarboxylic acid cycle (TCA or Krebs cycle)
0836422883 - VN DO NOT FORWARD GENETICS215
