1
Q
Describe the need for organisms to respire
A
- Respiration- process that occurs in living cells- releases he energy stored in organic molecules e.g. glucose
- The energy is immediately used to synthesise molecules of ATP from ADP and inorganic phosphate (Pi)
- ATP in cells can be hydrolysed to release energy needed to drive biological processes
- Microorganisms (eukaryotic and prokaryotic), plants, animals, fungi and Protoctists all respire to obtain energy
2
Q
Describe why living organisms need energy
A
- Energy is the capacity to do work
- Potential energy- The energy that is stored in complex organic molecules- e.g. fats, carbohydrates and proteins
- This is also chemical energy converted from light energy during photosynthesis
- When this energy is released from organic molecules via respiration, it can be used to make ATP to drive biological processes
3
Q
List biological processes that are driven by ATP
A
- Active transport
- Endocytosis
- Exocytosis
- DNA replication
- Cell division
- Movement- e.g. of bacterial flagella, eukaryotic cilia, undulipodia, motor proteins
- Activation of chemicals- glucose is phosphorylated at the beginning of respiration so that it becomes more reactive and able to be broken down to release more energy
4
Q
Describe 2 different types of metabolic reactions
A
- Anabolic- large molecules synthesised from smaller molecules
- Catabolic- hydrolysis of large molecules to smaller ones
5
Q
What type of energy do atoms and ions have (in living cells)
A
Kinetic energy
6
Q
Describe kinetic energy in ions/atoms
A
Kinetic energy allows them to move e.g. when molecules diffuse down a concentration gradient, moving from one place to another- use their kinetic energy
7
Q
Energy transfer between and within living organisms diagram
A
8
Q
Describe the role of ATP
A
- Standard intermediary between energy-releasing and energy-consuming metabolic reactions in both eukaryotic and prokaryotic cells
- energy currency- can be hydrolysed to ADP- releases phosphate- releases 30KJ of energy- used in metabolic reactions
9
Q
Describe the structure of ATP
A
- Phosphorylated nucleotide
- Each molecule consists of:
- adenosine (nitrogenous base adenine + the 5-carbon sugar ribose)
- 3 phosphate (phosphoryl) groups
- Phosphodiester bond between sugar and phosphate
- Glycosidic bond between sugar and nitrogenous base
- Phosphoanhydride bond between phosphates
10
Q
Full names of ATP, ADP and AMP
A
ATP- adenosine triphosphate
ADP- adenosine diphosphate
AMP- adenosine monophosphate
11
Q
Describe the stability and movement of ATP
A
- ATP is relatively stable (doesn’t break don into ADP and Pi) when in solution in cells
- However, it is readily hydrolysed by enzyme catalysis
- Whilst in solution, it can easily be moved from place to place within a cell
- Each cell requires the structures associated with respiration as it cannot cross the plasma membrane
12
Q
Describe the reactions associated with ATP
A
- Hydrolysis of ATP into ADP and Pi releases energy (requires water, catalysed by enzymes called ATPases)
- This reaction is coupled with an energy-consuming metabolic reaction- Condensation- ADP and P- releases water
- ATP is the immediate energy source for a metabolic reaction
13
Q
Is ATP advantageous over direct energy transfer from glucose, why
A
- yes
- When ATP is hydrolysed to ADP and P, a small quantity of energy is released for use in the cells - Cells can therefore obtain the energy they need for a process in small manageable amounts that will not cause damage or be wasteful
- ATP is referred to as the universal energy currency- occurs in all living cells and is a source of energy that can be used by cells in small amounts
14
Q
What is released in respiration and ATP hydrolysis, describe this
A
- Heat
- Not wasteful- helps keep living organisms ‘warm’ and enables their enzyme-catalysed reactions to proceed at or near their optimum rate
15
Q
Describe the amount of energy released through each hydrolysis reaction of ATP
A
- ATP –> ADP = 30.5 kJmol-1
- ADP –> AMP = 30.5 kJmol-1
- AMP –> Adenosine = 13.8 kJmol-1
- Total = 74.8
16
Q
List the 4 main processes involved in aerobic respiraton
A
- Glycolysis
- Link reaction
- Krebs Cycle
- Oxidative Phosphorylation
Last three only take place under aerobic conditions- the pyruvate molecules from glycolysis are actively transported into the mitochondria for the link reaction
17
Q
List the main processes involved with anaerobic respiration
A
- glycolysis
- Pyruvate is converted, in the cytoplasm, to lactate or ethanol
- In the process, the reduced NAMD molecules are reoxidised so that glycolysis can continue to run, generating 2 molecules of ATP for every glucose molecule metabolised
18
Q
Stages of respiration diagram
A
19
Q
Describe the use of enzymes in respiration
A
- Each stage is catalysed by a specific enzyme
- Reactions in respiration are examples of oxidation and reduction reactions
o Oxidation: loss of electrons (loss of hydrogen).
o Reduction: gain of electrons (gain of hydrogen).
20
Q
Describe the use of Coenzymes in respiration
A
Coenzymes are needed to assist other enzymes in a reduction or oxidation reaction (because they can pick up and lose hydrogen atoms)
Co-enzymes used in respiration:
- NAD- Nicotinamide Adenine Dinucleotide
- CoA- Coenzyme A
- FAD - Flavine Adenine Dinucleotide
Co-enzymes that have been reduced are used in the final stage of respiration (oxidative phosphorylation) which produces ATP
21
Q
Outline glycolysis
A
- biochemical pathway that occurs in the cytoplasm of all living organisms that respire (incl. many prokaryotes)
- involves sequnece of 10 reactions each catalysed by different enzyme
- some involve help of coenzyme NAD
- doesn’t require oxygen
- energy investment and energy pay off stage
- occurs in cytoplasm
22
Q
Describe NAD
A
- Nicotinamide adenine dinucleotide
- non-protein molecule
- helps dehydrogenase enzymes carry out oxidation reactions
- oxidised substrate molecules during glycolysis, the link reaction and the Krebs cycle
23
Q
Describe the synthesis/structure of NAD
A
Synthesised in living cells from:
- nicotinamide (vitamin B3)
- Ribose (5-carbon sugar)
- 2 x phosphoryl groups
24
Q
Describe the working of NAD
A
- the nicotinamide ring can accept 2 hydrogen atoms, becoming reduced NAD
- Reduced NAD carries the protons and electrons to the cristae of mitochondria and delivers them to be sed in oxidative phosphorylation for the generation of ATP from ADP and Pi
- When reduced NAD gives up the portions and electrons that it accepted during one of the first 3 stages of respiration, it becomes oxidised and can be reused to oxidise more substrate- in the process becoming reduced again
25
Outline the 3 main stages of glycolysis
1- Phosphorylation of glucose to hexose bisphosphate
2- Splitting each hexose bisphosphate molecule into 2 triose phosphate molecules
3- Oxidation of triose phosphate to pyruvate
26
Describe the first stage of glycolysis
Phosphorylation of glucose to hexose bisphosphate:
- glucose = hexose sugar (contains 6 carbon atoms)
- molecules are stable- need to be activated before they can split into two 3-carbon compounds
1) One molecule of ATP is hydrolysed (to ADP and Pi), the released phosphoryl group is added to glucose to make hexose monophosphate (fructose 1 phosphate)
2) Another molecule of ATP is hydrolysed (to ADP and Pi), the phosphoryl group is added to the hexose phosphate to form molecule of hexose bisphosphate
Products- one molecule of hexose bisphosphate that has 2 phosphate groups (one at C1 and other at C6)
The energy from the hydrolysed ATP molecules activates the hexose sugar, prevents it from being transported outside of the cell
27
Describe the second stage of Glycolysis
Splitting each hexose bisphosphate:
- Each molecule of hexose bisphosphate is split into two 3-carbon molecules- triose phosphate
- each still has phosphate group attached
28
Describe part 1 of the 3rd stage of glycolysis
Before oxidation- Second phosphate (from cytoplasm) gets added to each triose phosphate to form triose bisphosphate
Oxidation of triose phosphate:
- Dehydrogenase enzymes, aided by coenzyme NAD, remove hydrogen from each triose phosphate (oxidation of substrate as it is losing hydrogen atoms)
- 2 molecules of NAD accept the hydrogen atoms (protons and electrons)- become reduced
- 2 molecules of NAD are reduced for every molecule of glucose undergoing this process
29
Describe part 2 of the 3rd stage of glycolysis
Removal of phosphate group:
- subtsrate level phosphorylation
- 4 molecules of ATP are made for every 2 triose bisphosphate molecules undergoing oxidation
- Each phosphoryl group from the two triose phosphates is used to make 2 molecules of ATP- 4 total
- phosphorylation of ADP is condensation reaction- endogonic- energy transferred from substrate to ATP molecule
- products- 2 x pyruvate molecules
30
Describe the products of glycolysis
- 2 Molecules of ATP (4 were made in second part of stage three, but 2 were used for phosphorylation in stage 1- [4-2=2 net total])
- 2 molecules of reduced NAD
- 2 molecules of pyruvate (a 3-carbon sugar)
31
Why do we only have a low amount of ATP in our body at any one time
- only have around 5g at any time
- however, may used between 36 and 50kg each day
- possibly because the ATP molecules are continually being hydrolysed and then resynthesised
- at rest, a person consumes and continually generates ATP at the rate of 1.5kg per hour (increases when active)
32
Describe what happens to the pyruvates after being produced in glycolysis
* transported across the outer and inner mitochondrial membranes via a specific pyruvate H= symport- transport protein that transports two ions/molecules in same direction into the matrix
* Pyruvate then converted into 2C acetyl group (link reaction), acetyl group oxidised In Krebs cycle
33
Describe the link reaction
1) Carboxyl group is removed- decarboxylated (this is the origin of some of the carbon dioxide produced during respiration)
2) It is also dehydrogenated- hydrogen removed from pyruvate
3) Decarboxylation of pyruvate together with dehydrogenation produces an acetyl group
4) The acetyl group combined with coenzyme A (coA) to become acetyl CoA
5) The coenzyme NAD becomes reduced (accepts hydrogen from dehydrogenation)
34
Link reaction equation
2 pyruvate + 2NAD + 2CoA --> 2CO2 + 2 reduced NAD + 2 acetyl CoA
35
What happens after the link reaction
Coenzyme A accepts the acetyl group and, in the form of acetyl CoA, carries the acetyl group onto the Krebs cycle
36
Briefly outline the Krebs cycle
- Series of enzyme-catalysed reactions that oxidise the acetate from the link reaction to 2 molecules of carbon dioxide
- Conserves energy by reducing coenzymes NAD and FAD (Flaine adenine dinucleotide)
- Reduced coenzymes then carry the hydrogen atoms to the electron transport chain on the cristae
- Aerobic- not directly used in link/Krebs, but will not occur in absence of oxygen
37
Stages of the Krebs cycle with diagram
1:
- The acetyl group (released from acetyl CoA) combine with a 4C compound oxaloacetate
- Forms 6C compound citrate
2:
- Citrate is decarboxylated and dehydrogenated
- Produces:
* 5C compound
* One molecule of carbon dioxide
* One molecule of reduced NAD
3:
- This 5 C compound is further decarboxylated and dehydrogenated
- Produces:
* 4C compound
* One molecule of carbon dioxide
* One molecule of reduced NAD
4:
- 4C compound combined temporarily with, and is then released from, coenzyme A
- Substrate level phosphorylation takes place
- produces 1 molecule of ATP
5:
- The 4C compound is dehydrogenated
- Produces:
* Different 4C compound
* Molecule of reduced FAD
6:
- Rearrangement of the atoms in the 4C molecule- catalysed by an isomerase enzyme
- Followed by further dehydrogenation
- Regenerates a molecule of oxaloacetate so the cycle can continue
NB- For every molecule of glucose there are 2 turns of the Krebs cycle
38
Glycolysis- location, number of reduced NAD, Reduced FAD, Carbon dioxide and ATP (per glucose)
Location- Cytoplasm
NAD- 2
FAD- 0
CO2- 0
ATP- 2
39
The link recation- location, number of reduced NAD, Reduced FAD, Carbon dioxide and ATP (per glucose)
Location- Mitochondrial matrix
NAD- 2
FAD- 0
CO2- 2
ATP- 0
40
The Krebs cycle- location, number of reduced NAD, Reduced FAD, Carbon dioxide and ATP (per glucose)
Location- Mitochondrial matrix
NAD- 6
FAD- 2
CO2- 4
ATP- 2
41
Describe other substrates (besides glucose) that can be used in the Krebs cycle
* Fatty acids broken down into many molecules of acetate that enter the Krebs cycle via acetyl CoA
* Glycerol may be converted to pyruvate and enter the Krebs cycle via the link reaction
*Amino acids may be deaminated (Amino group NH2 is removed) and the rest of the molecule can enter the Krebs cycle directly (or be changed to pyruvate or acetyl CoA)
42
Describe the scientific investigation process leading to knowledge of the structure of the mitochondrion
- First identified in animal cells in 1840, and in plant cells in 1900
- Ultrastructure not worked out until 1950s- studies using electron microscopes
43
Overview of mitochondria structure including diagram
- Present in all eukaryotic cells
- May be rod-shaped, thread-like or spherical
- Diameters of 0.5-1 um
- Lengths of 2-5 um, occasionally up to 10 um
- Envelope consists of inner and outer phospholipid membrane
- Outer membrane is smooth
- Inner membrane folded into cristae- large surface area
- Between inner and outer mitochondria membranes of the envelope is an intermembrane space
- Mitochondrial matrix enclosed by inner membrane
44
Describe the structure of the inner membrane of the mitochondria
- has proteins that transport electrons
- protein channels associated with ATP synthase enzymes that allow protons to diffuse through them
45
Describe the structure of the mitochondrial matrix
- semi-rigid and gel-like
- contains mitochondrial ribosomes, looped mitochondrial DNA and enzymes for the link reaction and Krebs cycle
46
Describe how the structure of the mitochondrial matrix enables its function
Where the link reaction and Krebs cycle takes place
Contains:
- Enzymes that catalase the stages of these reactions
- Molecules of the coenzymes NAD and FAD
- Oxaloacetate (4C compound that accepts the acetyl group from the link reaction)
- Mitochondrial DNA (some of which codes for mitochondria enzymes and other proteins)
- Mitochondrial ribosomes, structurally similar to prokaryotic ribosomes, where these proteins are assembled
47
Describe how the structure of mitochondrias outer membrane enables its function
- The phospholipid composition of the outer membrane is similar to that of membranes and around other organelles in eukaryotic cells
- Contains proteins, some of which form channels or carriers that allow the passage of molecules, such as pyruvate, into the mitochondrion
48
Describe how the structure of mitochondrias inner membrane enables its function
- The lipid composition of the inner membrane differs from that of the outer membrane
- The lipid bilayer is less permeable to small ions such as hydrogen ions (protons) than is the outer membrane
- The folds (cristae) give a large surface area for the electron carriers and ATP synthase enzymes embedded in them
- The electron carriers are protein complexes arranged in electron transport chains (involved in oxidative phosphorylation)
49
Describe how the structure of the intermembrane space in the mitochondria enables its function
- Also involved in oxidative phosphorylation
- Inner membrane is in close contact with the mitochondrial matrix, so the molecules of reduced NAD and FAD can easily deliver hydrogens to the electron transport chain
50
Outline the final stage of respiration
- Oxidative phosphorylation- production of ATP in presence of oxygen
- Takes place in mitochondria
- Folded cristae give a large surface area for the electron carrier proteins and the ATP synthase enzymes
- Involves electron carrier proteins, arranged in chains called the electron transport chains, embedded in the inner mitochondrial membranes (the cristae), and a process called chemiosmosis
51
Describe what happens to NAD and FAD in the final stage of respirtaion
1) Reduced NAD and reduced FAD are re-oxidised when they deliver hydrogen atoms to the electron transport chain
2) The hydrogen atoms released from reduced NAD/FAD split into protons and electrons
3) The protons go into solution in the mitochondrial matrix
52
Describe the electron transport chain
- The electrons from the hydrogen atoms pass along the chain of electron carriers
- Each electron carrier protein has iron ion at its core
- The iron ions can gain an electron- becoming reduced (Fe2+)
- The reduced iron ion can the donate the electron to the iron ion in the next electron carrier in the chain, becoming re-oxidised to Fe3+
- Electron carrier proteins also contain oxido-reductase enzymes
- Electron carriers also have a coenzyme that, using energy released from the electrons, pump protons across the inner mitochondrial membrane, into the intermembrane space
53
Describe the proton gradient
- As protons accumulate in the intermembrane space, a proton gradient forms across the membrane
- Proton gradients generate a chemiosmotic potential that is also known as a proton motive force, pmf- they are a source of potential energy - ATP is made using the energy of the pmf
- Protons cannot easily diffuse through the lipid bilayer of the mitochondrial membranes, as the outer membrane has a low degree of permeability to protons and the inner membrane is impermeable to protons
- Protons can, however, diffuse through the protein channels associated with ATP synthase enzymes that are in the inner membrane
- ATP synthase enzymes are large and protrude from the inner membrane into the matrix
- As protons diffuse down their concentration gradient through these channels, the flow of protons causes a conformational (shape) change in the ATP synthase enzyme that allows ADP and Pi to combine, forming ATP
- This flow of protons is known as chemiosmosis- coupled to the formation of ATP
- Formation of ATP in presence of oxygen is oxidative phosphorylation
54
Describe the role of oxygen in the final stage of respiration
- Oxygen is final electron acceptor
- Combines with electrons coming off the electron transport chain
- Also combined with protons diffusing down ATP synthase channel
- Forms water
4H+ + 4e- + O2 --> 2H2O
55
Describe the quantity of ATP produced from oxidative phosphorylation
- The protons and electrons from the 10 molecules of NAD can theoretically produce 25 molecules of ATP
- The protons nd electrons from the 2 molecules of reduced FAD can theoreticalay produce 3 molecules of ATP
- Oxidative phosphorylation may therefore produce 28 molecules of ATP per molecule of glucose
56
Describe the ATP yield for each stage of aerobic respiration and the total
- Glycolysis
- Glycolysis- 2
- Link reaction- 0
- Krebs Cycle - 2
- Oxidative phosphorylation- 28
- Total- 32
57
Why is the theoretical yield of ATP rarely achieved in aerobic respiration
The theoretical yield is rarely achieved- actual yield may be closer to 30 ATP per glucose, or even less- because:
- Some ATP used to actively transport pyruvate into the mitochondria
- Some ATP used in a shuttle system that transports reduced NAD, made during glycolysis, into the mitochondria
- Some protons may leak out through the outer mitochondrial membrane
58
Describe what happens to respiration in the absence of oxygen
1) Oxygen can’t act as final electron acceptor at the end of oxidative phosphorylation- protons diffusing through channels associated with ATP synthase can’t combine with electrons and oxygen to form water
2) The concentration of protons in the matrix and reduces the proton gradient across the inner mitochondrial membrane
3) Oxidative phosphorylation ceases
4) Reduced NAD and reduced FAD are not able to unload their hydrogen atoms and cannot be reoxidised
5) The Krebs cycle and link reaction stops
59
Briefly outline the mechanism of anaerobic respiration
- For the organism to survive these averse conditions, glycolysis can take place, but the reduced NAD generated during the oxidation of triose phosphate to pyruvate has to be reoxidised so that glycolysis can continue
- These reduced coenzyme molecules cannot be reoxidised at the electron transport chain, so another metabolic pathway must operate to reoxidise them
60
Name the 2 metabolic pathways eukaryotic cells can use to reoxidise the reduced NAD, where do these occur
- Fungi (e.g. yeast) use the ethanol fermentation pathway
- Mammals use the lactate fermentation pathway
Both take place in the cytoplasm of cells
61
Describe the ethanol fermentation pathway
1) Each molecule of pyruvate (produced during glycolysis) is decarboxylated and converted to ethanal
o This stage is catalysed by pyruvate decarboxylase
o Has coenzyme thiamine diphosphate bound to it
2) The ethanal accepts hydrogen atoms from reduced NAD- becomes reduced to ethanol
o Enzyme ethanol dehydrogenase catalyses reaction
Reduced NAD is reoxidised and made available to accept more hydrogen atoms from triose phosphate- allows glycolysis to continue
62
Describe the lactate fermentation pathway
Occurs in mammalian muscle tissue during vigorous activity (e.g. running fast to escape predator) when demand for ATP for muscle contraction is high and there is an oxygen deficit.
1) Pyruvate (produced during glycolysis) accepts hydrogen atoms from the reduced NAD (also made during glycolysis)
o Enzyme lactate dehydrogenase catalyses the reaction
2 Outcomes:
- Pyruvate reduced to lactate
- Reduced NAD becomes reoxidised
The reoxidised NAD can accept more hydrogen atoms from triose phosphate during glycolysis, and glycolysis can continue to produce enough ATP to sustain muscle contraction for a short period
63
Describe the fate of lactate
- The lactate produced in the muscle tissue is carried away from the muscles, in the blood, to the liver
- When more oxygen is available, the lactate may be either:
* Converted to pyruvate- enter the Krebs cycle via the link reaction
* Reduced to glucose and glycogen
64
Describe the ATP yield from anaerobic respiration
- Neither ethanol fermentation nor lactate fermentation produces any ATP
- However, because this allows glycolysis to continue, the net gain of 2 molecules of ATP per molecule of glucose is still obtained
- Because the glucose is only partly broken down, many more molecules can undergo glycolysis per minute, and therefore the overall yield of ATP is quite large
- However, for each molecule of glucose, the yield of ATP via anaerobic respiration is about 1/15 of that produced by aerobic respiration
65
Hydrogen acceptor in lactate vs ethanol fermentation
- Lactate- pyruvate
- ethanol- ethanal
66
CO2 production in lactate vs ethanol fermentation
- lactate- no
- ethanal- yes
67
End product in in lactate vs ethanol fermentation
- lactate- lactate and NAD
- ethanol- ethanol- co2, NAD
68
Enzymes involved in lactate vs ethanol fermentation
- lactate- lactate dehydrogenase
- ethanol- pyruvate decarboxylase (w/ coenzyme thiamine diphosphate), ethanol dehydrogenase
69
What would happen if lactate were not removed from the muscle tissues
the pH would be lowered and this would inhibit the action of many of the enzymes involved in glycolysis and muscle contraction
70
List different substrates, describe
- carbohydrates
- lipids
- proteins
Can be oxidised in the presence of oxygen to produce molecules of ATP, carbon dioxide and water, each have different relative energy values
71
Describe carbohydrates as a respiratory substrate
- monosaccharide glucose is the chief respiratory substrate
- some mammalian cells such as brain and red blood cells can only use glucose for respiration
- animals and some bacteria store carbohydrates as glycogen, plants store as starch- can be hydrolized to glucose for respiration
- disaccharides can be digested to monosaccharides for respiration
- monosaccharides such as fructose and galactose can be changed, by isomerase enzymes, to glucose for respiration
72
Briefly outline lipids as respiratory substrates
Triglycerides hydrolysed by lipase to glycerol and 3 fatty acids
- glycerol can be converted to trio's phosphate and respired
- fatty acids (long-chain hydrocarbons) can go through beta-oxidation to reduce NAD/FAD and enter the Krebs cycle
NB- heart muscle can respire fatty acids
73
Describe how fatty acids can be used as a respiratory substrate
1) Use energy from hydrolysis of ATP (to AMP) to combine each fatty acid with coenzyme A
2) Fatty acid-CoA complex transported to mitochondrial matrix
3) broken down into several 2-carbon acetyl groups, each attached to CoA
4) This beta-oxidation pathway generates reduced NAD and FAD
5) The acetyl groups are released from CoA and enter the Krebs cycle by combining with 4C oxaloacetate
74
Describe how proteins can be used as a respiratory substrate
- excess amino acids (released after digestion of proteins) are deaminated in the liver
- converts into urea and keto acid
- the keto aid enters the respiratory pathway as private, acetyl CoA or a Krebs cycle acid e.g. oxaloacetic acid
- in fasting/starvation/prolonged exercise, protein from muscles can be hydrolysed into amino acids- converted to pyruvate or acetate and enter Krebs cycle
75
Respiratory substrates diagram
76
Describe why different respiratory substrates have different energy values
- most of ATP produced during aerobic inspiration is made during oxidative phosphorylation
- the greater the availability for chemiosmosis, the more ATP can be produced
- therefore, the more hydrogen atoms there are in a molecule of respiratory substrate, the more ATP can be generated per molecule of substrate
- as the protons (hydrogen ions) ultimately combine with oxygen atoms to from water the greater the proportion of hydrogen atoms in a molecule, the more oxygen will be needed for respiration
- e.g. fatty acids are long-chain hydrocarbons with a carboxylic acid group- many hydrogen atoms and very few oxygen atoms source of many protons
77
Mean energy value (kJg-1) of carbohydrate, lipid and protein
- carbohydrate- 15.8
- Lipid- 39.4
- Protein- 17.0
78
What is used to describe the energy value of different respiratory substrates
Respiratory quotient (RQ)
79
Respiratory quotient formula
CO2 produced / O2 consumed
80
Respiratory quotient of glucose, fatty acids and amino acids
81
Respiratory quotient values- interpretation
RQ of greater than one indicates some anaerobic respiration is taking place, as more CO2 is being produced than O2 being consumed
82
Describe yeast
- facultative anaerobe
- single-celled fungus
- eukaryotic (cells contain mitochondria)
- yeast cells may reproduce asexually by dividing by mitosis
83
Describe respiration in yeast
- if oxygen is available, he yeast cells respire aerobically using glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation- producing many molecules of ATP per glucose molecule
- if oxygen is lacking, they respire anaerobically rising glycolysis and the ethanol pathway- produces only a few molecules of ATP per molecule of glucose
84
Outline investigations in respiration in yeast
Aerobic vs anaerobic:
- for yeast cells to divide, they require ATP
- rate of reproduction depends on the amount of ATP available
- would expect yeast to have faster rate of reproduction under aerobic conditions
- yeast can oxidise ethanol under aerobic conditions
- alcohol content of cider is around 6%- not enough to kill the yeast
- with anaerobic respiration, this may increase, whereas it will decrease in the aerobic flasks
85
Outline investigating the rate of reproduction of yeast cells under aerobic and anaerobic conditions
- in the presence of oxygen, yeast cells can oxidise the ethanol and any sugar in the cider, and will not be killed by the alcohol - they will respire aerobically and produce more ATP that can be used for cell division
- under anaerobic conditions, the ethanol can't be oxidised and will eventually kill the yeast
- therefore, where the respiration is aerobic, more yeast cells should be present per cm3 after a week
86
Describe the use of different flask sizes in yeast respiration investigations
- in the small flasks, the cider is deep and with a small surface area for oxygen absorption and a large diffusing distance for oxygen to each of the yeast cells
- opposite true for large flasks
87
Nam of the equipment used to measure the number of cells in a sample
haemocytometer
88
Method of investigating the rate of reproduction of yeast cells under aerobic and anaerobic conditions
1) pour 50cm3 of cider into each of the conical flasks
2) using clean pipette, add one drop of yeast suspension to each conical flask
3) place 4 layers of muslin, cheesecloth or tights material over the mouth of each flask and secure with elastic band- allows oxygen to enter but keeps out dust and contaminants
4) leave in arm place for around a week
5) mix thoroughly by swirling, and using clean pipette, withdraw some of the flask contents and place a drop onto a haemocytometer slide with coverslip in place
6) count the number of yeast cells in centre square and each corner square- if on boundary, count only cells touching north and west side of square
7) this shows cells per 0.002mm3- muliply by 50000 to find number per cm3
8) repeat for 3 different sizes of flask- alters oxygenavailability
89
Precautions taken when investigating respiration in yeast under aerobic and anaerobic conditions
- conical flasks must be clean and sterilised
- cider should be uncontaminated
- swirl flask containing yeast suspension before removing
90
Describe haemocytometers
- each is special thick slide with bevelled edges and grooves
- thicker coverslips used- may be grooves in H shape- 2 etched grids- 2 counts an be made from same sample
91
Method of using a haemocytometer
1) Breathe onto underside of coverslip to moisten it
2) Slide the coverslip horizontally onto the slide and carefully press down with the index fingers whilst pushing with the thumbs
3) when the coverslip is correctly in position, you will see six rainbow patterns (newtons rings)- the depth of each chamber is now 0.1mm
4) place the pipette tip at the entrance to the groove and allow liquid to fill the chamber
5) leave for 5 mins before counting
6) place slide on microscope stage with one of the grids over the stage aperture
7) focus using total x40 magnification
8) focus using total x100 magnification
9) the central portion of the grid will now fill the field of view
10) count cells in the central and 4 corner squares
92
Describe other ways of comparing aerobic and anaerobic respiration in yeast
- the rate of respiration can be measured by measuring the create of evolution of carbon dioxide
- as carbon dioxide dissolves in the culture medium it lowers the pH, and this can be measured using a pH metre
93
Equipment used to measure rate of respiration
Respirometer
94
Describe the principle of respirometers
- organisms that are respiring aerobically absorb oxygen and give out carbon dioxide
- if the carbon dioxide produced is absorbed by sodium hydroxide solution or solid soda lime, then the only volume change within the respirometer is due to the volume of oxygen absorbed by the organisms
- if oxygen is absorbed from the tube containing the organisms, then that tube has a reduced volume of air in it, exerting less pressure than the greater volume of air in the other tub- as a result, the coloured liquid in the manometer tube rises up towards the respirometer tube
- if the original level of liquid in the manometer tube is marked and the radius of the bore in the capillary tube is known, the volume of oxygen absorbed during a specific period can be calculated
95
Describe how to reset respirometer apparatus
- syringe depressed to inject air into the system and reset the liquid in the manometer tube back to its original position
- this also allows a reading of the volume of oxygen absorbed by noting the change in level of the syringe plunger, as measured from the graduated scale on the syringe barrel
96
respirometer diagram
97
describe how to set up respirometer apparatus
1) after placing the coloured liquid, for example, methylene blue solution that has one drop of detergent added to it, into the manometer tube, the apparatus is connected with the taps open- enables the air in the apparatus to connect with the atmosphere
2) find mass of living organisms (e.g. woodlice)
3) with the taps still open the whole-set up, with the living organisms in place, is placed ina water bath for at least 10 mins until it reaches the temperature of the water bath
4) the syringe plunger should be near the top of the scale on the syringe barrel and its level noted
5) the levels of coloured liquid in the manometer tubes can be marked with felt tip of chinagraph pencil
6) the taps are closed and the apparatus is left in the water bath for a specific period, such as 10 mins
7) the change in the level of manometer liquid can be measured, and the syringe barrel depressed to reset the apparatus- also allows volume of oxygen absorbed to be measured
8) can then calculate the volume of oxygen absorbed per minute per gram of living organism
98
Describe how the effects of temperature can be measured using a respirometer
- readings taken at each temperature
- in between each reading, the apparatus and organisms should be allowed to adjust to the new temperature
- suitable living organisms can be blowfly maggots, woodlice, least in glucose suspension or soaked pea seeds that are beginning to germinate
- animal specimens should only be used over a narrow range of temperatures e.g. from 10 to 40 degrees
99
describe investigating the effect of substrate concentration on the rate of respiration in yeast
- the respirometer may be used so that a suspension of yeast, with differing concentrations of glucose solution, is placed in one of the tubes
- if the sodium hydroxide solution is omitted the evolution of CO2 during specific time period can be measured
100
Describe the investigation of different respiratory substrates in yeast
- yeast may be able to produce isomerase enzymes to change some types of monosaccharide to glucose so that glycolysis can take place
- yeast may also be able to produce enzymes to hydrolysed disaccharides to monosaccharides
- the respirometer, without sodium hydroxide solution, could be used to measure evolution of carbon dioxide within a specific period
101
Describe an alternative investigation of different respiratory substrates in yeast
- carbon dioxide produced during the anaerobic respiration of yeast can be collected in fermentation tubes
- the liquid in the fermentation tube is a solution of a particular sugar plus 2 drops of yeast suspension
- set up series of fermentation tubes, with same conditions except type of sugar (e.g. glucose, fructose and galactose)
- measure height of carbon dioxide bubble after set time period- can see how efficiently yeast desire different substrates
- could also try disaccharide sugars e.g. maltose, sucrose and lactose
