Bacterial Respiration, fermentation, growth Flashcards
What are the names of the 2 phases of Glycolysis?
Investment phase and Payout phase
In brief, what happens in the investment phase of glycolysis and how much ATP is spent?
6 carbon Glucose is cleaved into two 3 carbon Glyceraldehyde, 2 ATP is spent to phosphorylate during this phase.
In brief, what happens in the pay-out phase of glycolysis and how much ATP is made?
2 ATP is made for each Glyceraldehyde molecule converted to pyruvate , so per 1 Glucose molecule 4 ATP is made
What is the overall yield of a) ATP b) NADH per 1 glucose molecule of glycolysis?
a) 2 ATP (2 spent in investment phase, 4 made in pay-out phase)
b) 2 NADH (1 made per glyceraldehyde and there’s 2 per glucose molecule)
Why does glycolysis not require oxygen?
As ATP is produced by substrate-level phosphorylation not by an electron transport chain which requires a terminal electron acceptor.
What is substrate phosphorylation?
When a phosphate group is directly transferred to ADP.
Where does glycolysis occur in eukaryotic cells?
In the cytoplasm
What are the products of the link reaction?
One molecule of NADH is formed per pyruvate (so 2 NADH per glucose)
What is the process of the link reaction?
Pyruvate from glycolysis is decarboxylated to Acetyl CoA (releasing CO2 and NADH)
What is the yield of a) ATP b) NADH c) FADH2 produced from the Krebs cycle from one cycle and from 1 Glucose molecule?
a) Per 1 cycle: 1 ATP
>Per 1 Glucose molecule: 2 ATP
b) Per 1 cycle: 3 NADH
>Per 1 Glucose molecule: 6NADH
c) Per 1 cycle: FADH2
>Per 1 Glucose molecule: 2 FADH2
Why are there 2 cycles of the Krebs cycle per 1 glucose molecule?
There are 2 cycles per glucose molecule as 2 mol of pyruvate will be present from the link reaction.
What are the overall yields of a) ATP b) NADH c) FADH2 from glycolysis, link reaction, and Krebs cycle per 1 glucose molecule and what are they used for?
a) 4 ATP (2 from Krebs and Glycolysis)
>Stored as energy
b) 10 NADH (6 from Krebs, 2 from Link, 2 from Glycolysis)
>Used in ETC due to high reducing power (act as electron carrier molecules)
c) 2 FADH2 (both from Krebs)
>Used in ETC due to high reducing power (act as electron carrier molecules)
What are Quinones?
Quinones are lipophilic molecules (hydrophobic) found in membrane which carry electrons
How many electrons and protons are required to reduce a quinone to a quinol ?
2 Electrons and 2 Protons
What type of anaerobe are E.coli?
Facultative anaerobe
What does it mean that E.coli are facultative anaerobes?
Means if O2 is present does aerobic respiration (allows full oxidation of growth substrate, maximal conserved energy as O2 as terminal electron acceptor) but if it isn’t either anaerobic respiration or fermentation occur.
When would a) Aerobic respiration b) Anaerobic respiration c) Fermentation occur in E.coli?
a) In presence of oxygen
b) No oxygen, but in presence of alternative terminal electron acceptor
c) No oxygen or alternative terminal electron acceptor.
Where do both glycolysis and the Krebs cycle occur in E.coli and how does this differ to eukaryotic cells?
Both processes occur in the cytoplasm (different to eukaryotes as glycolysis occurs in the cytoplasm and Krebs in the mitochondria)
Where is the periplasm found?
Periplasm is between outer and inner membrane.
Where is the a) Positive (p/+) b) Energetic c) Negative (n/-) areas for the ETC in E.coli?
a) Periplasm is positive
b) Inner membrane is energetic membrane
c) Cytoplasm is negative
What is meant by the inner membrane being the “energetic membrane” in the ETC in E.coli?
Inner/ cytoplasmic membrane is energetic membrane, where the ETC is localised and the proton gradient/ proton motive force is built up to drive ATP synthesis.
In terms of the periplasm, inner/cytoplasmic membrane, and cytoplasm, what is the movement of H+ throughout the ETC in E.coli?
H+ pumped from cytoplasm (N side, contains less H+) to periplasm (P side, contains more H+), moves back through to cytoplasm via ATP synthase in inner membrane (down conc gradient)
What are NADH dehydrogenase complex I and Succinate dehydrogenase complex II examples of?
Electron donor complexes in eukaryotes.
What are the electron donor complexes alternatives to a) Complex I (NADH dehydrogenases) b) Complex II (succinate dehydrogenase) in E.coli?
a) Nuo and Ndh (NADH dehydrogenases)
b) SDH (succinate dehydrogenase)
Is there an equivalence to complex III or cytochrome c in E.coli?
No
What is found in E.coli instead of Cytochrome c oxidases and what is their function.
> 2 terminal quinol oxidases called Cyo and Cyd
> Directly oxidise quinols to quinones releasing electrons to convert oxygen to water.
Where do electrons enter the Nuo complex and where do they leave?
Electrons are transferred from NADH to the FMN cofactor in Nuo, travels through 9-Fe-S clusters where the electron transfers to reduce quinones.
Which NADH dehydrogenase also acts as a proton pump in E.colI?
Nuo
How does Nuo pump protons from the N side (cytoplasm) to the P side (periplasm)?
Large membrane domain contains 4 proton channels, the oxidation of NADH and reducing quinones to quinols provides energy to pump 4 protons from N to P.
How is Ndh similar to Nuo and different?
> Similar as is a NADH dehydrogenase, so oxidises NADH and reduces quinone to quinols by uptake of protons
> Different as Ndh is monotopic (only interacts with one side of membrane), so can’t act as a proton pump from N to P side.
How does Succinate Dehydrogenase (SDH) mediate quinone to quinol reduction
Succinate oxidised to fumarate, electrons diffuses and reduces FADH2, then donates electrons to quinones to form quinols and returns to FAD.
Why can succinate dehydrogenase (SDH) not act as a proton pump from N side to P side?
There is not as much energy released from succinate oxidation compared to NADH so not enough energy to couple these reactions to move protons
What do all Nuo, Ndh, and SDH require to reduce Quinones to Quinols?
Uptake of protons from cytoplasmic (N) side (from NADH or FADH oxidation).
What do electron donor complexes in E.coli do?
Use protons and electrons to reduce Quinones to Quinols.
What do terminal oxidases in E.coli do?
Oxidise Quinols to Quinones to release electrons.
What are the 3 electron donor complexes in E.coli?
Nuo, Ndh, SDH
What are the 2 terminal quinol oxidases in E.coli
Cyo and Cyd
How does coupling a donor complex and an acceptor complex generate a proton motive force?
Generates a REDOX loop: The reduction of quinones to quinols using protons on N side via donor complexes allows acceptor complexes to oxidise quinols releasing protons onto P side creating a proton motive force (indirectly moves protons through membrane)
Which quinol oxidase has a higher proton: electron ratio and why?
Cyo has a higher H+/e- (4:2, two electrons moves 4 protons to P side) than Cyd (2:2) because in addition to the indirect movement of 2 protons via the REDOX loop, Cyo also directly pumps 2 protons from N to P.
Which quinol oxidase works under a) Hyperoxic (high O2 conc) b) microoxic (low O2 conc) conditions and why?
a) Cyo, as has low O2 affinity
b) Cyd, as has high O2 affinity
Why is Cyd useful for pathogenicity in 2 reasons?
- Works under low O2 conc conditions due to high affinity
- More resistant to sulphide, hydrogen peroxide, nitric oxide (found in gut)
What combination of donor and acceptor complex gives the highest Proton:Electron ratio in E.coli and how much ATP does this produce per NADH oxidised when oxygen is the terminal electron acceptor?
Nuo and Cyo moves 8 protons from N->P per 2 electrons (4:1 H+/e-), therefore can produce 2.4 ATP per NADH oxidised (8/3.33= 2.4 ATP)
How many protons does it take to produce 1 ATP via ATP synthase in E.coli?
3.33 H+ per ATP, so 10 protons per 3 ATP.
Under high oxygen conditions, what combination of electron donor and electron acceptor is used in E.coli and when is this advantageous?
Ndh and Cyo are used despite lower proton:electron ratio (as aren’t proton pumps). It has a quicker turnover rate so can produce NAD+ quicker. Advantageous if bacteria is in highly reduced area as needs to oxidise NADH to NAD+ quickly.
What is advantageous about E.coli being able to swap combinations of electron acceptors and donors?
Using different combinations of these complexes, the bacteria can alter the PMF produced depending on the environment they are found in.
What is a) Nuo b) Ndh more favourable for in bacteria and why?
a) Maximal energy efficiency due to high proton:elecron ratio.
b) Increased metabolic flux/ growth rate due to quicker turnover of NAD+
Why are Ndh and Cyd good targets for antimicrobials?
As they are present in many pathogenic bacteria (used for reacting to environmental change) while not being present in mitochondria.
What are 4 ways to measure bacterial growth?
- Cell dry weight
>Measure change in mass of a culture (measures dead cells too) - Cell number
>Total count= counts live and dead cells
>Viable count= counts just live cells - Optical density (most common)
>Is an indirect measure so requires a standard curve to relate the optical density values to the cell number. - Specific cell component
>Measure a specific cell component as an indirect measurement of growth.
Describe a batch culture
A culture with a fixed volume (closed system).
How do bacterial cells divide?
Binary fission
What does division by binary fission allow?
Leads to exponential growth
What is the time taken for a generation to double called?
Generation time or doubling time, this varies between different organisms.
What 2 factors cause variation in generation time?
- The specific organism
- The environmental conditions.
Why is unrestrained growth in batch culture not possible in 2 reasons?
- As it’s a closed system the essential nutrients become totally depleted
- Metabolism leads to an accumulation of end products leading to autoinhibition, as toxins excreted cannot be removed, the change in pH slows growth.
Describe the 4 different phases of batch culture growth?
- Lag phase
>Bacteria preparing for growth - Log phase
>Once cells adapt to new environment, bacteria start to grow and divide by binary fission (exponential / logarithmic growth)
>Nothing limits growth - Stationary phase
>When nutrients run out or toxins build up, cells remain metabolically active waiting for favourable conditions to occur.
>Growth and death balances out. - Death phase
>Without input of nutrients into media or without removing toxic compounds, over time leads to exponential death
Describe how the Lag phase can vary in length for 1) cells moved from media to a similar media 2) cells moved from rich or not rich media to a new media?
- If we take cells from a media and moved to a similar media the lag phase is short as they don’t need long to adjust to their surroundings and grow exponentially again.
- But if we take cells from a rich or a not rich media and put it in a new media, they need to adjust to growing under new conditions more by synthesizing new enzymes making long phase longer.
What scale is needed for an exponential growth graph?
Need Log scale on Y axis to make straight line for exponential growth
How do you work out the increase in cell number during exponential growth period if starting with 1 cell?
1*2^number of generations
(1 is the number of cells started with)
What is the same and different between aerobic and anaerobic respiration in E.coli?
For both processes E.coli use membrane-embedded electron-transport chain to generate a PMF to produce ATP but for anaerobic they use a terminal acceptor other than oxygen
What are 5 examples of alternative electron acceptors used by E.coli during anaerobic respiration and their Midpoint potentials (mV)?
- Nitrate +360mV
- Nitrite +420mV
- Fumarate +30mV
- Dimethyl +160V
- Trimethylamine N-oxide +130mV
Describe a thermodynamically favourable electron transfer
Electron donors have a more negative redox potential (or Midpoint potential) than electron acceptors (electron traveling to more and more positive REDOX/ Midpoint potentials), so electrons flow through these chains and release energy to drive proton motive force.
What are the 2 Quinone and Quinol types E.coli uses and their Midpoint potentials (mV)?
- Ubiquinone/ Ubiquinol for aerobic conditions as more positive potential (UQ/UQH2 +110 mV)
- Menaquinone/ Menaquinol for anaerobic as more negative potential (MK/MKH2 -75 mV)
What is midpoint potential and what is it measured in?
> Midpoint potential is measured in milli volts (mV)
> A measure of the tendency of a compound to take electrons from other compounds.
What 1) Electron donors 2) Quinone/ Quinol 3) Electron acceptors do E.coli use in aerobic conditions and their midpoint potentials?
1) NADH/NAD+ (-340mV)
>Succinate/ fumarate (+30mV)
2) Ubiquinone/ Ubiquinol (UQ/UQH2 +110 mV)
3) O2 (+820mV)
Was is oxygen the best electron acceptor?
As it has a very high midpoint potential (+820mV), so electrons release a lot of energy when transferred to it.
What electron donors are used by E.coli in anaerobic conditions and what is their Midpoint potentials?
- Formate (-430mV)
- H+ (-420mV)
- NADH (-340mV)
- Lactate (-190mV)
- Gly-3-P (-190mV)
What electron donors are used by E.coli in anaerobic conditions and what is their Midpoint potentials?
- Formate (-430mV)
- H+ (-420mV)
- NADH (-340mV)
- Lactate (-190mV)
- Gly-3-P (-190mV)
What Quinone/ Quinol are used for anaerobic conditions and their Midpoint Potentials?
Menaquinone/ Menaquinol (MK/MKH2 -75 mV)
What is the difference between Glycolysis in aerobic and anaerobic conditions and why?
Glycolysis is the same in aerobic and anaerobic producing 2 pyruvate molecules as it doesn’t require oxygen.
What is the difference between Glycolysis in aerobic and anaerobic conditions and why?
Glycolysis is the same in aerobic and anaerobic producing 2 pyruvate molecules as it doesn’t require oxygen.
What happens to Pyruvate produced by glycolysis in aerobic conditions?
Pyruvate is decarboxylated by Pyruvate dehydrogenase to produce Acetyl Co (link reaction)
What is meant by a) catabolism b) anabolism?
a) Metabolic pathways and biochemical reactions in living organisms that break down complex molecules into simpler ones
b) Set of metabolic pathways and biochemical reactions in living organisms that build complex molecules from simpler ones
Why is the Krebs cycle referred to as amphobolic.
As it is used for both catabolism and anabolism.
What are the a) catabolic b) anabolic functions of the Krebs cycle in E.coli?
a) Catabolic function in producing NADH and FADH (reduced electron carrier)
b) Metabolic functions:
>Citrate (catabolic) for Fatty acids (anabolic/ biosynthesis)
>Alpha-Ketoglutarate for Glutamate, other amino acids, purines
>Succinyl-CoA for Hemes
>Oxaloacetate for Aspartate, other amino acids, Purines, pyrimidines
How is ATP produced from Acetyl-CoA in anaerobic conditions?
> Under anaerobic conditions PDH (pyruvate-dehydrogenase complex) is inhibited and pyruvate is converted to formate and acetyl-CoA by pyruvate formate lyase (PFL)
> Acetyl-CoA is converted to by phospho-transacetylase and acetate kinase acetate generating ATP by substrate-level phosphorylation
When is formate produced and what is its role in E.coli?
> Pyruvate is converted into formate and acetyl-CoA by pyruvate formate lyase (PFL)
> Formate can act as an electron donor to the anaerobic ETC via the formate dehydrogenase enzyme (FDH)
When oxygen returns to conditions, how is substrate level phosphorylation from Acetyl CoA inhibited?
PFL is inhibited by oxygen – switch back to pyruvate decarboxylation by PDH (pyruvate-dehydrogenase complex) under aerobic conditions
What are the 2 branches of the Krebs cycle under anaerobic conditions?
- Oxidative branch
>Ends at alpha-Ketoglutarate as without presence of O2, alpha-Ketoglutarate dehydrogenase enzyme is inhibited. Used for glutamate and other amino acids. - Reductive branch
> If PEP (precursor of pyruvate at end of glycolysis) is carboxylate to form oxaloacetate (still used as precursor for amino acids), goes down this pathway
>Fumarate is made and this can act as an electron acceptor to be reduced to succinate.
What is the difference between aerobic and anaerobic Krebs cycle in E.coli?
> Aerobic= Is a cycle
> Anaerobic = consists of 2 branches (Oxidative and Reductive branches).
Other than use in the Krebs cycle, in anaerobic conditions what happens to the majority of Acetyl-CoA and why?
Most pyruvate is converted to acetate and secreted from the cell as an overflow metabolite to maintain REDOX balance.
Is NADH still produced by glycolysis in anaerobic conditions?
Yes when Glucose is converted to pyruvate as it doesn’t require oxygen.
What are used as electron donors in anaerobic conditions?
NADH (from glycolysis) and Formate (from pyruvate by PFL enzyme) can be used as electron donors for ETC If a suitable electron acceptor is present
If there is no suitable electron acceptor present while in anaerobic conditions what happens?
If there is no suitable electron acceptor present, fermentation occurs.
Describe an example ETC under anaerobic conditions where MK/MKH2 (Menaquinone/ Menaquinol) are switched to and why, also how much ATP is made?
- Electron donor
>NADH/NAD+ (-340mV), Nuo is the favoured NADH hydrogenase as has higher proton:electron ratio - Quinone/Quinol
>Menaquinone/ Menaquinol (-75mV), switches to these due to succinate having such a low positive Midpoint potential. - Succinate/ Fumarate (+30mV)
> 1.2 ATP per NADH (electron donor) oxidised for NADH to fumarate / 2 to 1 proton to electron ratio.
Why do E.coli have such a range of electron donors and acceptors, as well as Quinones/quinols for anaerobic and aerobic respiration?
As their environments are constantly changing, they must be able to use a range of molecules to allow respiration in varying conditions.
In anaerobic conditions with Fumarate as a terminal electron acceptor, which electron donor complexes are used and why?
- This uses Nuo instead of Ndh, as Nuo translocates protons from N side to P side (while Ndh doesn’t).
- This uses Frd instead of Cyo there is no point having a proton pump as Fumarate reduction to Succinate does not produce release enough energy for proton translocation.
What is the most efficient ETC that can be used under anaerobic conditions?
- Electron donor
>Formate/CO2 -430 mV, very good electron donor - Quinone/ Quinol
>Menaquinone/ Menaquinol (-75mV) - Electron acceptor
>Nitrate (NO2-/NO3-) +420mV, best terminal acceptor other than O2.
How are nitrates always used over all other electron acceptors in anaerobic conditions?
When nitrate is present it represses ither proteins for other acceptors.
In anaerobic conditions of Formate -> Nitrate, which electron donor complexes are used and why and the proton:electron ratio?
- Formate dehydrogenase (Fdn)
>As catalytic domain faces the periplasm, the 2 protons released from oxidation of Formate are released onto the P side. - Nitrate reductase (Nar)
>Protons from Quinol oxidation are released into the periplasm. Catalytic domain in cytoplasm is energy conserving as the 2 protons released from MK oxidation are not used to reduce Nitrate to Nitrite on the cytoplasmic side (can be reused/ REDOX loop) but instead H+ which are already on the N side.
> 2:1 proton to electron ratio (per 2 electrons, 4 protons are translocated to the periplasm).
Why is a Fdn and NaP worse for Formate -> Nitrite (anaerobic conditions) than Fdn and Nar?
> As NaP’s catalytic domain is in periplasm, the enzyme is not energy conserving as protons released from MK oxidation (into P side) are cancelled out because the reduction of Nitrate to Nitrite is done on the P side so 2H+ are used up on that side anyway. (while Nar has a catalytic domain on the N side so this doesn’t happen).
> Leads to 1.2ATP produced per Formate oxidised for Fdn+NaP which is half of what Fdn+Nar produced (1.2ATP per).
Despite being less energy conserving due to catalytic domain on P side, what are 2 reasons why E.coli would use NaP over Nar?
- Nap can function at lower nitrate conditions than Nar
- Nap doesn’t require the uptake of a negatively charged nitrate ion into the cytoplasm, it is done in the periplasm
How do bacteria decide which Electron donor complexes to use?
Bacteria balance proton motive force generation with other considerations based off of the demands of the cell and the conditions it is found in
When would fermentation occur?
If no oxygen, and no alternative electron acceptor.
What is used as an electron acceptor during fermentation?
Fermentations use endogenous organic molecules as electron acceptors in the absence of oxygen and a respiratory electron transport chain
How is ATP produced during fermentation?
ATP production is limited to substrate-level phosphorylation in the cytoplasm
Why is glycolysis necessary for fermentation?
Glycolysis (initial breakdown of sugar molecule) generates pyruvate which acts as the organic electron acceptor and the NADH produced is used to reduce Pyruvate.
What would stop glycolysis from occurring?
If NADH+ is not re-oxidised (e.g. by ETC) then glycolysis will stop.
Why is glycolysis referred to as an incomplete catabolic pathway?
As the NADH+ generated must be re-oxidised (e.g. by ETC) in order for glycolysis to occur again (as needs NAD so it can be reduced).
Wy does fermentation lead to less biomass produced than aerobic respiration?
As produces many side products (e.g. CO2 and Hydrogen) which are excreted.
What is an advantage of fermentation side products being excreted?
Hydrogen, for example, then can act as an electron donor to other organisms.
Why are so many waste products secreted by fermentation?
As ATP yields are very low (1-3 mol ATP per mole of glucose) so many quick rounds of fermentation are needed to produce the same amount of ATP as aerobic respiration; these quick rounds produce a lot of side product which needs to be excreted.
What are 3 human areas which fermentation is useful in?
- Economic
- Biotechnological
- Cultural importance (e.g. alcohol)
What happens to the Pyruvate produced from glycolysis during fermentation and how is this used for substrate phosphorylation?
> In the absence of oxygen pyruvate remains in the cytoplasm and is reduced to lactate using NADH as electron donor
> This regenerates NAD+ and restores redox balance allowing glycolysis to continue to make ATP by substrate-level phosphorylation (substrate phosphorylation = glucose -> Pyruvate producing 2NADH + 2ATP)
What are the 6 types of fermentation and what is the difference between them?
- Homolactic fermentation
>Glucose (or another sugar) is converted into lactic acid by a single enzymatic reaction. - Heterotactic fermentation
>Glucose is metabolized through a series of enzymatic reactions that produce multiple end products, including lactic acid, ethanol, and/or acetic acid. - Alcohol fermentation
>glucose is converted to ethanol and carbon dioxide - Mixed acid fermentation
> glucose is metabolized by bacteria to produce a mixture of organic acids, including acetic acid, lactic acid, and formic acid - Acetone-Butanol-Ethanol (ABE) fermentation
>glucose is metabolized by certain bacteria to produce a mixture of solvents, including acetone, butanol, and ethanol. - Metabolic fermentation
> can refer to any type of fermentation process that involves the metabolic conversion of organic compounds by microorganisms.
Describe Homolactic fermentation
- Glycolysis: Glucose converted to 2 Pyruvate (requires 2NAD+ reduced to 2NADH)
- 2 Pyruvate converted to 2 Lactate by Lactate dehydrogenase (2NADH oxidised to 2NAD+)
- 2NAD+ can be re-used by Glycolysis where glucose is converted to 2 Pyruvate and as this produces 2 ATP (substrate level phosphorylation).
What type of bacteria carry out Homolactic and Heterotactic fermentation?
Lactic acid bacteria (e.g., certain species of Lactobacillus)
Describe Heterotactic fermentation and what are the products?
> Glycolysis is broken down by Phosphoketolase pathway
- Rather than splitting glucose into 2 Pyruvate (3C), make Ribulose-5-phosphate (5C, one carbon is lost as a CO2),
- isomerised into different 5 carbon sugar, this is split into Glyceraldehyde-3-phosphate (3C) and Acetyl-P (2c) by Phosphoketolase
- Glyceraldehyde is metabolised to Pyruvate (NAD+ reduced to NADH producing 2ATP) then Lactate dehydrogenase converts to Lactate (NADH oxidised to NAD+)
- Acetyl-P is converted to ethanol by 2 dehydrogenase steps (each oxidising NADH to NAD+, producing 2NAD+ overall)
The net products are per glucose: 1 lactate, 1 ethanol, 1 CO2, 1ATP
Describe Alcoholic (ethanol) fermentation and its products
- Glucose converted to 2 Pyruvate (2NAD+ reduced to NADH, producing 2 ATP)
- 2 Pyruvate decarboxylated by Pyruvate decarboxylase to produce 2 Acetaldehyde (released 2CO2)
- 2 Acetaldehyde converted to 2 Ethanol by Alcohol dehydrogenase (oxidises 2NADH to 2NAD+).
- 2NAD+ can be used again for glucose -> Pyruvate producing 2 ATP per glucose via substrate level phosphorylation
What causes a REDOX balance during fermentation?
The regeneration of NAD+ by oxidising NADH so that glycolysis can occur over and over to produce 2 ATP (glucose -> 2 Pyruvate).
What microorganisms carry out alcohol fermentation?
Carried out by yeast (e.g. Saccharomyces cerevisiae) and some bacteria (e.g. Zymomonas mobilis unwanted contaminant in alcohol)
How do E.coli do fermentation?
By mixed acid fermentation
What is different about the mixed acid fermentation?
As there are so many different end products which can be made (Ethanol, acetate, Formate, succinate, Hydrogen, carbon dioxide), the proportions of the end products vary depending on growth conditions to balance ATP production and redox balance (compare to other fermentations where product stoichiometry is fixed)
What are some of the end products produced by mixed acid fermentation?
Ethanol, acetate, Formate, succinate, Hydrogen, carbon dioxide (some like Formate and Hydrogen can be secreted as waste products and can be taken up by neighbouring cells as an alternative electron acceptor).
Describe mixed acid fermentation and the products
- Glucose -> 2 Pyruvate by glycolysis
- Pyruvate-Formate lyase enzyme is induced under anaerobic conditions, splits Pyruvate into 2Acetyl-CoA and 2 Formate
- Some Acetyl-CoA can go into anaerobic Krebs branches, some is converted to Acetate producing ATP (acetate then excreted), or converted to Ethanol producing two NAD+
- Formate (if terminal electron acceptor is present is used as an electron donor) is converted to hydrogen and CO2 (can diffuse out of the cell so Formate doesn’t build up as is an acid)
> Overall production: 1 Ethanol, 1 Acetate, 2H2 + 2CO2 all get excreted, 3 ATP (2 from glycolysis, 1 from acetate formation), REDOX balance from 2 NADH oxidised to 2NAD+ from Ethanol production.
How are a) Succinate b) Lactate also produced by mixed acid fermentation in some conditions?
a) If PEP or pyruvate is converted to oxaloacetate in the reductive branch of anaerobic Krebs
b) Pyruvate converted to lactate via pyruvate dehydrogenase
What is an advantage of Fumarate and Succinates production by mixed acid fermentation in E.coli?
Formate can act as an electron donor while Succinate can act as an electron acceptor, so can carry out some anaerobic respiration by ETC even in fermentation conditions.
What is the order of ATP yield out of fermentation, anaerobic respiration, aerobic respiration?
- Aerobic produces the most
- Anaerobic produces the second most
- Fermentation produces the least as not ETC is used (only ATP produced via substrate level phosphorylation).
Which type of fermentation is important for industry and why?
Acetone-Butanol-Ethanol (ABE) fermentation for producing butanol as a renewable biofuel.
What is Acetone-Butanol-Ethanol (ABE) fermentation carried out by?
Carried out by Gram positive Clostridium species (e.g., Clostridium acetobutylicum)
What are the 3 products of Acetone-Butanol-Ethanol (ABE) fermentation and in what ratio?
3:6:1 acetone:butanol:ethanol
Why is Malolactic fermentation referred to as secondary fermentation?
Occurs after a microorganism has done a primary fermentation. E.g. for wine making, yeast fermentation occurs first to make alcohol.
Describe Malolactic fermentation (MLF) used for wine production
- Produce alcohol by Yeast based fermentation
- Malic acid (present in wine) taken up into cell, converted into lactate and lactate is excreted by the anti-porter which takes in the Malic acid
- As Malate (malic acid) has a charge of -2 and Lactate of -1, the anti-port generates a membrane potential (takes in 2 negative charges from outside and only excreting 1) by using a proton to convert Malate to lactate
- Generates a proton motive force to produce ATP (chemiosmotic) so doesn’t require REDOX balancing.
What are the disadvantages to batch culture?
- Isn’t accurate representation of bacteria growth in their actual niche, as is a closed system.
- If comparing two batch cultures, must compare at a point of the same cell density for example, as the conditions change throughout the batch culture (due to product secretion changing pH) so is must take measurements at the same points for valid comparison.
- Can’t examine microbes under nutrient limited conditions as they grow at maximum rate in batch culture.
What is an advantage to batch culture in the lab?
Useful for comparing WT and mutant strains
What are 2 examples of continuous culture systems, what is similar and different about them?
- Chemostats
>Fresh medium is continuously added - Turdistats
>Fresh medium is periodically added
For both the spent culture (including cells) is siphoned away (so volume stays the same but substrates needed for growth are replenished while toxic side produce is removed).
What are the 5 advantages to continuous (open systems) culture
a) adding substrates/nutrients for growth
>So new substrates are added for growth, growth can occur for longer.
b) autoinhibitory products are diluted
>toxic side products are removed so growth can occur for longer
c) bacterial populations can be maintained in exponential phase at a constant cell density
>As in batch culture cell density increases in a closed system, in continuous we can match the growth rate of the culture to the amount of media added (keep cell density constant)
d) growth rate and cell density can be controlled independently
>Can independently control growth rate and density (can have the same growth rate at different cell densities and vice versa), so can measure submaximal growth rates at different growth limitations.
e) In steady state, the growth of organisms can be studied under precisely controlled physiochemical conditions
What is the main difference between continuous culture and batch culture?
Continuous culture is an open system (media is added and spent culture is removed during growth) while batch culture is a closed system (media is neither added or removed).
What does steady state mean and which system can do this?
> When culture conditions remain virtually constant.
> Continuous/ open system cultures can maintain this by adding and removing media.
What are similarities between chemostats and turbidostats?
- Culture volume constant
>Media added and spent culture removed is at the same volume - Must be well mixed
>So medium is well balanced, keeping uniform distribution of cells and nutrients as well as keeping oxygen tension constant. - pH kept within pre-determined limits
- Temperature kept constant
>Both pH and temperature effect growth rates - Stability affected by wall growth and foaming >use of silanising agents and antifoam chemicals, as bacteria sticking to walls, precipitate (froth, foam) and mutate (effecting how they grow) changes the steady state.
What are 4 defining features of Chemostat cultures?
- Fresh medium is supplied at a constant rate (the flow rate/dilution rate is constant) and spent culture is removed at the same constant rate, thus the culture volume remains constant (different to periodical addition in turbidostats).
- The growth medium contains a limiting concentration of one essential nutrient (others in excess) -
>prevents maximum growth rate (growth limiting nutrient) (e.g. carbon-limited Chemostat) - Can control growth rate over a wide submaximal range
>Can increase dilution rate to increase growth rate - Can control growth rate and population density independently of the other
>Can keep flow rate the same but can increase substrate concentration so steady state stays the same but biomass increases
How is steady state reached in a Chemostat culture in 3 steps?
- Growth rate > dilution rate (cell number increases)
>Initially not many cells, so conc of growth limiting substrate is less growth limiting (enough substrate for small number of cells) - Growth rate < dilution rate (cell number decreases)
>As cells increase, the conc of growth limiting substrate decreases, so growth rate is slower than dilution so more cells are loss from removal of media than are grown. - Growth rate = dilution rate (steady state)
>Cycle keeps happening until an equilibrium between growth rate of bacteria and rate of removal of old culture is the same.
What are the properties of a Chemostat culture at steady state?
Growth rate is equal to dilution rate at this point (so have fixed doubling time and fixed cell density and conc of limiting substrate conc is constant)
In a chemostat culture, how is a) bacterial conc kept constant over a different range of growth rates b) keep growth rates the same but have different bacterial concentrations?
a) If we increase dilution rate, the limiting nutrient conc remains low and constant (despite adding more substrate, the cells use this to grow a little faster) the bacterial concentration remains the same despite changing growth rates/ doubling time going down
b) If we add more of a growth limiting substrate at a set dilution rate (doesn’t change growth rate), the bacterial conc would increase (increase steady state cell density, can decrease it if remove growth limiting substrate)
In a chemostat culture what two variables can be changed independently of one another?
- Can keep bacterial conc (cell density) the same over different growth rates.
- Can keep constant growth rates but have different bacterial conc (cell density)
These both can be changed independently of each other.
Why does steady state chemostat break down at low dilution rates?
> At low dilution rates bacterial concentration is decreased due to the requirement of maintenance energy (substrates used for other processes than growth).
> At low dilution rates, as less substrate is fed in the total consumed substrate used for cell maintenance is higher compared to that used for growth so higher cell densities are not reached at low dilution rates (as substrate levels are so low, a higher proportion is used for maintenance energy instead of growth).
What is maintenances energy?
Maintenance energy is the use of the growth-limiting substrate for essential cellular functions other than growth (e.g. protein synthesis)
Why does steady state chemostat break down at high dilution rates (above critical dilution rate)?
If dilution rate is higher than growth rate of bacteria, then bacteria are washed out at a quicker rate than they can divide. Bacterial concentration decreases and limiting nutrient increasing is a sign of this as there is no bacteria left to use it up.
What is a Turbidostat culture
A continuous culture with a growth-dependent feedback system, in which the dilution rate is controlled by monitoring cell density (Turbidity)
How are different cell densities set in a Turbidostat culture?
> Is a dynamic response at cell density (not a constant flow of media like chemostat)
> Turbity (set by cell density) is maintained at a constant level by spectrometer detecting turbidity levels: if turbidity is above set desired value, dilution rate increases (new media added and same volume of old culture removed) keeping cell density constant.
What are 2 differences between Chemostat and Tubrbidostat cultures?
- Turbidostat increases dilution rate when spectrometry measures an increased turbidity (increased cell density), like a negative feedback mechanism, to keep cell density constant.
>Chemostat has continuous dilution flow to reach steady state. - Turbidostat contains no-growth limiting substrate, so cells grow at a maximum rate at a constant population density under constant conditions.
>Chemostat contains growth-limiting substrate, so can alter bacterial concentration and growth rates independent of each other at steady state.
What is industrial microbiology and describe a common property of its products?
> Industrial microbiology is the large scale production of commercial products by microorganisms (mostly they already make the product without genetic engineering).
> Here the products are often relatively low value but we want very high yields
What is industrial microbiology and describe a common property of its products?
> In microbial biotechnology microbes are engineered to produce non-native compounds (e.g. insulin, somatotropin)
> Biotechnological products are typically high value and are produced in lesser quantities than in traditional industrial microbiology
What are 5 examples of microbial products and what produces them?
- Antibiotics e.g. penicillin (Pencillium chrysogenum), tetracycline (Streptomyces spp.)
- Enzymes e.g. lipases (Candida cylindraceae), amylases (Bacillus subtilis), lactase (Kluyveromyces lactis)
- Food additives e.g. vitamins (e.g. riboflavin; Ashbya gossypii, Bacillus subtilis), amino acids (Corynebacterium glutamicum)
- Chemicals e.g. citric acid (Aspergillus niger), bioethanol (Saccharomyces cerevisiae), butanediols (E. coli)
- Terpenes e.g. artemisinin (Saccharomyces cerevisiae), carotenoids
- Alcoholic drinks e.g. beer, wine, spirits (yeasts)
What are 8 useful properties of industrial microbes?
- They produce the substance of interest (in high yield)
>High proportional of cell mass used to make product - Grow rapidly (and reproducibly) and produce product in relatively short period of time
- Can grow and form product in large scale culture under bioreactor conditions
- Grow in simple and inexpensive growth media without complex nutrient and vitamin requirements
- Metabolic flexibility/adaptability
>Can use cheaper feedstock. - Do not produce toxins/toxic by-products and are not pathogenic to humans, animals or plants
- Amenable to genetic engineering and are genetically stable
>Sequenced genome so we can genetically engineer microbe
>Genetically stable, so don’t genetically mutate while making stock - Can be stocked or stored
>Some microbes not easy to store long term in cryostock. - They secrete the product into media (bonus) or are easy to break/handle
What does fermentation refer to in industrial settings?
In industrial settings, fermentation refers to growth of large quantities of cells (the fermenters) within a vessel called a fermenter (or bioreactor) for production of commodity chemicals, biofuels, pharmaceuticals, enzymes, etc (most industrial fermentations are actually aerobic)
What is a key process to get a culture from a lab to an industrial level?
Scaling up (increase lab culture to industrial scale).
What are the three methods for industrial fermentation and which is most commonly used in industry?
batch, fed-batch (most commonly used) or continuous culture.
Describe industrial batch fermentation
In batch fermentations, all of the nutrients required for the fermentation are provided in the initial culture medium. Once these nutrients have been consumed, growth of the organism ceases and the fermentation is ended.
Describe industrial continuous fermentation
Continuous fermentations are performed by continually supplying fresh medium to the culture with the subsequent removal of the same amount of culture, resulting in a steady state being reached in the fermenter.
Describe industrial fed-batch fermentations and why is it most commonly used in industry?
> Nutrients provided initially for a batch culture medium, allowing for exponential growth (generating biomass). Once nutrients start to run out (stationary phase), can feed culture with more nutrients producing high levels of product
> As produces the most product.
Describe Penicillium chrysogenum fed-batch fermentation for penicillin production in 5 steps (example of a fed-batch fermentation)
- Initial growth phase in a small fermenter inoculated with freeze-dried spores
- Scaled up through two further growth stages in successively larger fermenters to provide a large enough inoculum for the production phase
- The fermentation production phase is a fed-batch culture with high oxygen levels maintained and Carbohydrates? and Nitrogen feeding
- Carefully monitored to keep the fermentation in optimal penicillin production mode during production phase, which lasts for 120 to 200 hours
- Penicillin is excreted into the medium and is recovered at the end of the fermentation
What are 5 methods to improve product yield?
- Random Mutation and selection of one which causes production at a higher yield.
- Metabolic engineering/synthetic biology
- Nutritional/physiological approaches (Different carbon sources fed)
- Optimising fermentation conditions (type of bioreactor)
- More than one/all of the above (In combination can drastically increase yield)
If we know the pathway for how a microbe creates a desired product but the microbe isn’t suitable for commercial process, what can be done?
Can transfer this pathway into a bacterium that is suitable for commercial process
What is Bioprospecting?
Bioprospecting is the search for organisms, enzymes or natural products with potential commercial applications (Often looking for extremophiles as can survive commercial conditions).
What is Metagenomics?
Recover Nucleic acid from specific environments and introduce DNA to bacteria as plasmids. Can be made into a library.
What is gene mining?
Gene mining is the process of identifying and isolating genes from environmental samples without having to culture to the organism
What is the definition of metabolic engineering and what is its goal?
> Metabolic engineering is the deliberate redesign of cellular biochemical pathways to enhance production of a product or to produce a novel product
> Goal: re-direct cellular metabolism to make more of desired products and minimise unwanted products while not negatively effecting growth of host.
What must be balanced while genetically modifying bacteria?
Balance growth and stability of engineered strain with the maximum produced product.
What are 6 ways genetic engineering can be done to increase product yields?
- Modifying the metabolic pathway to redirect existing metabolism to specific products
>Look at branch points where product can make 2 substrates and we want one - Enhancing the precursor and energy/cofactor supply to the pathway by engineering central metabolism
>E.g. increasing ATP and NADH needed - Engineering transport systems
>Maybe needed if microbe doesn’t normally use this pathway, and helps recover product as it is excreted. - Increasing cellular tolerance to the product or substrate
>If the substrate is toxic the microbe at high conc - Consideration of regulatory effects such as product feedback inhibition
>Products feedback to earlier enzyme in pathway to stop production of the products when it has sufficient amounts, but we want more. - Decoupling of growth and product formation
>Useful if product is toxic, so we grow biomass first then produce lots of product
Why is genetic engineering important despite only increasing product yields by small %?
Even small improvements can make a big difference to the economics of the process
What is an example of 1) Improving the yield of a natural product 2) Introducing a complex biosynthetic pathway into a new chassis?
1) lysine production by Corynebacterium glutamicum
2) vitamin B12 production in E. coli
What bacteria produces and secretes Lysine?
Corynebacterium glutamicum (aerobic and gram positive bacterium).
What us Lysine?
An amino acid we cannot make, so need to ingest.
What method is used to produce lysine on an industrial scale?
Fed-batch fermentation of Corynebacterium glutamicum
What is fed to Corynebacterium glutamicum to produce lysine and what type of pathway is it?
> Glucose
> Branched pathway
Describe the 4 genetic engineering methods for increasing lysine production by Corynebacterium glutamicum
- Elimination of allosteric feedback-inhibition using anti-metabolites
>Lysine and Threonine production leads to allosteric feedback to inhibit aspartate kinase (LysC) (needs to be a combination of both products binding to LysC).
>Add anti-metabolite (e.g. aminoethyl-L-cysteine) to allosterically inhibit LysC, these also show the variants of an enzyme which don’t undergo allosteric inhibition so can mutate beta subunit of LysC to prevent Lysine binding allowing unlimited Lysine production. - Promotor engineering to improve metabolic flux at the aspartate semi-aldehyde branch point
>Increase chance that the Aspartate semi-aldehyde intermediate produces lysine, by overexpressing the naturally present DapA (dihydrodipicolinate synthase) by point mutations (first enzyme in Lysine branch)
>1.3 fold increase in lysine production - Increasing cofactor supply by over-production of transhydrogenase
>Lysine production involves NADPH oxidation, over-production of lysine causes REDOX imbalance (need more NADPH)
>Introduce Transhydrogenase (PntAB): Interconverts the reduced NADH and oxidised NADP+ cofactors to reduced NADP and oxidised NAD+. As Cellular NADH pool greater than cellular NADPH pool and we make a a lot of NADP+ by producing lysine - overexpression of pntAB regenerates NADPH levels allowing for overproduction of Lysine - Increasing Lysine secretion by over-producing the lysine exporter LysC
>Overexpress LysE (naturally present lysine exporter) to excrete Lysine so doens’t reach toxic high conc
>But has to be controlled so not too much as would cause damage to cell membrane.
Describe a simplified version of the branched Lysine pathway
- Oxaloacetate
- Aspartate (LysC converts 2 -> 3)
- Aspartate semi-aldehyde, branched pathway:
a. Lysine
b. Eventually makes other amino acids
(Methionine, Threonine or Isoleucine)
What type of enzyme is LysC and what is its role in Lysine production?
LysC (aspartate kinase) converts Aspartate to Aspartate semi-aldehyde.
How does an anti-metabolite like aminoethyl-L-cysteine inhibit an enzyme?
It is a substrate analogue to an allosteric inhibitor, but it doesn’t inhibit the enzyme once bound and isn’t broken down, so the original substrate cannot inhibit the enzyme allosterically anymore (e.g. Lysine to LysC)
How many molecules of NADP are needed for production of 1 mol of Lysine?
4 molecules of NADPH (oxidised to NADP+)
What are the 2 ways Lyse E (Lysine exporter) removes lysine from Corynebacterium glutamicum?
As Lysine is positively charged at neutral pH active export requires either:
- Symport: 2OH- (two hydroxyl ions) brings Lys out the cell with it
- Antiport: using proton gradient into the cell pushing Lysine out
What is Vitamin B12 and what does deficiency cause?
> Vitamin B12 (cobalamin) is one of the eight B vitamins and plays an essential role as a coenzyme in animals
> Only synthesised by some prokaryotes, humans must consume it, severe deficiency can lead to pernicious anaemia
What precursor is used for vitamin B12 and briefly describe the pathway
> Pathway branches from the universal tetrapyrrole precursor uroporphyrinogen III
> Very complicated, needing 25 enzymes.
Why doesn’t E.coli naturally produce vitamin B12?
As it is expensive to make (needs 25 enzymes) so is easier to find from environment.
What are 5 steps to increase product yield of Vitamin B12 from E.coli while maintaining their optimal growth?
- Optimised expression of genes
- Enhanced the uptake and chelation of cobalt
- Increased metabolic flux to the uroporphyrinogen III starting substrate
- Downregulated the competing heme and siroheme biosynthesis pathways
- Optimised the fermentation process
