Unit 4 Objectives

Question 1

Describe anabolism

Answer 1

A metabolic process where simple molecules are combined to form complex molecules, utilizing energy in the process.

Question 2

Describe catabolism

Answer 2

A metabolic process that involves the breakdown of complex molecules into simpler ones, releasing energy in the form of ATP in the process.

Question 3

Describe glycolysis

Answer 3

The metabolic process that oxidizes glucose to make 2 pyruvate producing a net gain of 2ATP and 2 NADH in the cytoplasm of the cell

Question 4

Describe enzymes

Answer 4

Biological catalysts, typically proteins, that accelerate chemical reactions in living organisms by lowering the activation energy required for the reaction to occur

Question 5

Describe fermentation

Answer 5

An anaerobic metabolic where organisms, such as yeast and certain bacteria, convert sugars (like glucose) into byproducts, such as alcohol, acids, and gases. Energy is released and used to regenerate NAD+ from NADH

Question 6

Describe reduction-oxidation (redox)

Answer 6

Reduction-oxidation (redox) a chemical reaction that involves transfer of electrons between two substances. In a redox reaction, one substance is oxidized (loses electrons) while another is reduced (gains electrons)

Question 7

Describe aerobic respiration

Answer 7

A metabolic process in which organisms convert glucose and oxygen into energy, carbon dioxide, and water. This process occurs in three main stages:

Glycolysis
Krebs Cycle (Citric Acid Cycle)
&
Electron Transport Chain (ETC):

Question 8

Describe oxidative phosphorylation

Answer 8

The final stage of aerobic respiration, occurring in the inner mitochondrial membrane, where ATP is produced through the coupling of electron transport and chemiosmosis

Electron Transport Chain (ETC)
&
Chemiosmosis

Question 9

Describe photosynthesis

Answer 9

A biochemical process used by green plants, algae, and some bacteria to convert light energy into chemical energy in the form of glucose, using carbon dioxide and water as raw materials. This process occurs primarily in the chloroplasts of plant cells and can be divided into two main stages:

Light Reactions: when light is absorbed by chlorophyll and other pigments, it energizes electrons, which are then transferred through a series of proteins in the electron transport chain. Generates ATP and NADPH while splitting water molecules, releasing oxygen as a byproduct.
&
Calvin Cycle (Light-Independent Reactions)

Question 10

Describe chemiosmosis

Answer 10

Chemiosmosis is a process by which ATP is produced in cells by using the energy generated from the movement of protons (H⁺ ions) across a membrane.

This occurs:
during oxidative phosphorylation
and
during photophosphorylation

using a:
Proton Gradient Formation
&
ATP Synthesis

Question 11

Describe final electron acceptor

Answer 11

A substance that receives electrons at the end of an electron transport chain (ETC) during cellular respiration and certain types of fermentation.

Allows flow of electrons to continue through the ETC, facilitating the generation of ATP.

In Aerobic Respiration: the final electron acceptor is molecular oxygen (O₂)

In Anaerobic Respiration: the final electron acceptor can be substances other than oxygen, such as nitrate (NO₃⁻), sulfate (SO₄²⁻), or carbon dioxide (CO₂)

In Fermentation: the final electron acceptor is often an organic molecule.
For instance, in alcoholic fermentation: acetaldehyde

In lactic acid fermentation: pyruvate

Question 12

Describe photophosphorylation

Answer 12

The process of ATP from ADP and inorganic phosphate (Pi) using the energy derived from sunlight.

Occurs in the chloroplasts of plant cells during photosynthesis

two main types:
non-cyclic photophosphorylation
&
cyclic photophosphorylation

Question 13

Describe substrate-level phosphorylation

Answer 13

ATP is produced through the direct transfer of a phosphate group from a phosphorylated substrate to ADP.

This mechanism contrasts with oxidative phosphorylation, where ATP is generated through an electron transport chain and a proton gradient

Question 14

Briefly describe what happens in glycolysis (also known as Embden-Meyerhof pathway)

Answer 14

Glycolysis is the first step in the breakdown of glucose to extract energy for cellular metabolism. It occurs in the cytoplasm of the cell and consists of ten enzyme-catalyzed reactions. Glycolysis converts 1 molecule of glucose (a 6-carbon sugar) into 2 molecules of pyruvate (a 3-carbon compound), producing a net gain of 2 ATP and 2 NADH molecules.

Question 15

Include the description of the 3 stages of glycolysis? (Stage 1)

Answer 15

1. Energy Investment Phase (Preparatory Phase):
   
   • Steps 1-3: Two molecules of ATP are consumed to phosphorylate glucose, which is then split into two 3-carbon sugar phosphates.

Glucose is first converted to glucose-6-phosphate and then to fructose-1,6-bisphosphate. This prepares glucose for further breakdown and traps it inside the cell.

Question 16

Include the description of the 3 stages of glycolysis? (Stage 2)

Answer 16

2. Cleavage Phase (Splitting Stage):
   
   • Step 4: Fructose-1,6-bisphosphate is cleaved by the enzyme aldolase into two 3-carbon molecules: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).

DHAP is then converted into another G3P, so two G3P molecules continue through the rest of glycolysis.

Question 17

Include the description of the 3 stages of glycolysis? (Stage 3)

Answer 17

3. Energy Payoff Phase (Payoff Stage):
   
   • Steps 5-10: The two G3P molecules undergo a series of reactions that produce 4 ATP and 2 NADH molecules (via substrate-level phosphorylation and redox reactions).

In the final step, pyruvate is formed. Since 2 ATP molecules were used in the first stage, the net yield is 2 ATP and 2 NADH.

Question 18

What organisms is Glycolysis for?

Answer 18

Glycolysis provides quick energy and can occur in the absence of oxygen, making it a vital metabolic pathway for both aerobic and anaerobic organisms

Question 19

Briefly describe what happens in aerobic respiration (cellular respiration)

Answer 19

Aerobic respiration (cellular respiration) is the process by which cells convert glucose into ATP, using oxygen as the final electron acceptor.

It consists of three main stages: the transition step, the Krebs cycle (TCA cycle), and the electron transport chain (ETC).

In total, aerobic respiration produces up to 38 ATP per glucose molecule: 2 from glycolysis, 2 from the Krebs cycle, and about 34 from the ETC

Question 20

Include description of the transition step

Answer 20

1. Transition Step (Pyruvate Oxidation):

After glycolysis, the two pyruvate molecules are transported into the mitochondria (or the cytoplasm in prokaryotes).

Each pyruvate is converted into acetyl-CoA by the enzyme pyruvate dehydrogenase, releasing one molecule of carbon dioxide (CO2) and generating NADH.

This step prepares the acetyl-CoA for entry into the Krebs cycle.

Question 21

Description of Krebs (TCA) cycle

Answer 21

2. Krebs Cycle (TCA Cycle):

Acetyl-CoA enters the cycle and combines with oxaloacetate to form citrate.

Through a series of reactions, citrate is broken down, releasing two molecules of CO2 per cycle and generating 3 NADH, 1 FADH2, and 1 ATP (or GTP) for each turn of the cycle.

Since two acetyl-CoA molecules are produced from one glucose molecule, the Krebs cycle runs twice, producing a total of 6 NADH, 2 FADH2, 2 ATP, and 4 CO2.

Question 22

Description of the electron transport chain

Answer 22

3. Electron Transport Chain (ETC) and Oxidative Phosphorylation:

Electrons pass through a series of protein complexes, releasing energy that pumps protons (H+) across the membrane, creating a proton gradient.

Proton gradient powers ATP synthase, which generates ATP from ADP through chemiosmosis.

Oxygen acts as the final electron acceptor, combining with electrons and protons to form H2O

Located in the inner mitochondrial membrane (or

plasma membrane in prokaryotes

Yields around 34 ATP molecules per glucose molecule

32 for prokaryotes

Question 23

Briefly describe Fermentation

Answer 23

Fermentation is an anaerobic process that allows cells to generate energy when oxygen is not available. It occurs after glycolysis and helps regenerate NAD+ so that glycolysis can continue to produce ATP.

There are two main types of fermentation:

lactic acid fermentation
&
alcoholic fermentation

Question 24

Briefly describe Lactic acid Fermentation

Answer 24

1. Lactic Acid Fermentation:
   • After glycolysis, pyruvate is reduced to lactic acid by the enzyme lactate dehydrogenase, using electrons from NADH.
   • This process regenerates NAD+ so glycolysis can continue.
   • produce yogurt and other fermented foods.
   • Net gain: 2 ATP per glucose (from glycolysis)

Question 25

Briefly describe Alcohol Fermentation

Answer 25

2. Alcoholic Fermentation:
   • After glycolysis, pyruvate is first converted into acetaldehyde and releases CO2
   • Acetaldehyde is then reduced by NADH to form ethanol.
   • This process also regenerates NAD+, for glycolysis
   • It occurs in yeast and some bacteria, used in the production of alcoholic beverages and bread.
   • Net gain: 2 ATP per glucose (from glycolysis)

Question 26

Briefly describe lipid catabolism

Answer 26

Lipid catabolism is the process of breaking down lipids (fats) to generate energy. This occurs primarily through the breakdown of triglycerides into glycerol and fatty acids

1. Glycerol Breakdown:
   
   • Glycerol is converted into (G3P)
2. Fatty Acid Oxidation (Beta-Oxidation):
   
   • Fatty acids are broken down into acetyl-CoA units

Question 27

Why does Lipid catabolism yield more ATP than carbohydrate breakdown?

Answer 27

Due to the higher energy content of fats

Question 28

Briefly describe protein catabolism

Answer 28

Protein catabolism is the process of breaking down proteins into their individual amino acids for energy or other cellular processes.

1. Deamination:
   
   • Amino acids are stripped of their amino group (NH2), generates ammonia (NH3) and a carbon skeleton.
   • The amino group is often converted into urea (in humans) and excreted.
2. Carbon Skeleton Metabolism:
   
   • The remaining carbon skeletons are converted into intermediates that enter the Krebs cycle, or glycolysis, such as pyruvate, acetyl-CoA, or oxaloacetate.

Question 29

When does protein catabolism occur?

Answer 29

Protein catabolism generally occurs when carbohydrates and fats are not available as primary energy sources

Question 30

Briefly describe the photosynthetic process

Answer 30

Photosynthesis is the process by which plants, algae, and some bacteria convert light energy into chemical energy stored in the form of glucose

It takes place in two main stages:

light-dependent reactions
&
the Calvin cycle

Question 31

Describe light-dependent reactions

Answer 31

1. Light-Dependent Reactions:

Occur in the thylakoid membranes of chloroplasts.

Light energy is absorbed by chlorophyll and used to split water molecules, releasing O2, electrons, and protons.

The electrons move through the electron transport chain, generating ATP and reducing NADP+ to NADPH.

This stage produces ATP, NADPH, and O2.

Question 32

Describe the Calvin cycle

Answer 32

2. Calvin Cycle (Light-Independent Reactions):

Occurs in the stroma of chloroplasts.

The ATP and NADPH produced in the light-dependent reactions are used to fix CO2 into organic molecules, ultimately forming glucose.

The key enzyme, RuBisCO, helps convert CO2 into a 3-carbon molecule that eventually leads to the production of glucose.

Question 33

Describe cyclic photophosphorylation

Answer 33

Cyclic Photophosphorylation:

In this process, electrons from photosystem I are recycled back to the electron transport chain, generating ATP only (no NADPH or O2 produced).

Question 34

Describe non-cyclic photophosphorylation

Answer 34

Noncyclic Photophosphorylation:

Electrons flow from photosystem II to photosystem I, and then to NADP+, producing both ATP and NADPH. Oxygen is released as a byproduct.

Question 35

Briefly describe anaerobic respiration.

This is different from fermentation.

Answer 35

Anaerobic respiration is a type of cellular respiration that occurs in the absence of oxygen, using an alternative final electron acceptor instead of oxygen.

Unlike fermentation, anaerobic respiration uses the ETC to generate ATP, final electron acceptor, nitrate, sulfate, or CO2.

1. Glycolysis
2. Krebs Cycle
3. ETC

Question 36

How is Anaerobic respiration different than fermentation?

Answer 36

Anaerobic respiration produces more ATP than fermentation

Anaerobic respiration uses an ETC with a final electron acceptor other than oxygen

Fermentation relies on substrate-level phosphorylation to generate ATP.

Question 37

Describe the two alternatives to glycolysis

Answer 37

The pentose phosphate pathway (PPP): a metabolic pathway that generates NADPH and ribose-5-phosphate for biosynthesis

&
The Entner-Doudoroff pathway (EDP): a degradation pathway found in some bacteria that produces ATP, NADPH, and pyruvate

Question 38

Compare pentose phosphate pathway (PPP) to glycolysis

Answer 38

The PPP focuses on producing NADPH and R5P for biosynthesis

While glycolysis is primarily geared toward generating ATP and pyruvate for energy

Question 39

Compare Entner-Duodoroff (EDP) pathway to glycolysis

Answer 39

The EDP produces less ATP (1 ATP per glucose) than glycolysis (2 ATP per glucose)

but generates both NADPH and NADH, making it useful for both energy production and biosynthesis

Question 40

In prokaryotes, compare & contrast (glycolysis + fermentation) and (aerobic respiration) include theoretical net ATP yield, location in cell, & end products.

Answer 40

Glycolysis + Fermentation:
- Theoretical Net ATP Yield:
- 2 ATP per glucose molecule (from glycolysis).

• Location in Cell:
  
  • cytoplasm
• End Products:
  
  • Varies by type of fermentation;
    includes lactic acid or ethanol (lactic) and CO2 (alcoholic), along with the regeneration of NAD+ for glycolysis

Aerobic Respiration:
- Theoretical Net ATP Yield:
- Up to 38 ATP per glucose molecule (including ATP from glycolysis, Krebs cycle, and ETC).

• Location in Cell:
  
  • Glycolysis occurs in: cytoplasm
    -Krebs cycle & ETC occurs in: cytoplasmic membrane (as prokaryotes lack mitochondria).
• End Products:
  
  • Produces CO2 & H2O
    along with high-energy electron carriers (NADH and FADH2) utilized in the electron transport chain.

Question 41

Compare & contrast glycolysis + aerobic respiration in prokaryotes and eukaryotes include theoretical net ATP yield, location in cell, & end products.

Answer 41

Glycolysis:

Prokaryotes:
- Theoretical Net ATP Yield: 2 ATP per glucose molecule (from substrate-level phosphorylation).
- Location in Cell: cytoplasm
- End Products: Pyruvate, NADH

Eukaryotes:
Theoretical Net ATP Yield: 2 ATP per glucose molecule (same as in prokaryotes).
- Location in Cell: cytoplasm
- End Products: Pyruvate, NADH

Aerobic Respiration:

Prokaryotes:
- Theoretical Net ATP Yield: Up to 38 ATP per glucose molecule (including ATP from glycolysis, Krebs cycle, & ETC)
- Location in Cell:
- Glycolysis: cytoplasm
- Krebs cycle: cytoplasm
- ETC: cytoplasmic membrane
- End Products: CO2, H2O, NADH & FADH2

Eukaryotes:
- Theoretical Net ATP Yield: Up to 36 ATP per glucose molecule (2 from glycolysis, 2 from Krebs cycle, and approximately 32 from ETC).
Location in Cell:
- Glycolysis: cytoplasm
Krebs cycle: mitochondrial matrix
ETC: inner mitochondrial membrane
- End Products:
- CO2, H2O, NADH & FADH2

Question 42

What is true about prokaryotes & aerobic respiration ATP?

Answer 42

Aerobic respiration can yield slightly more ATP in prokaryotes due to a more direct ETC without the need to transport carriers into mitochondria in eukaryotes.

Question 43

Compare & contrast lactic acid fermentation and alcohol fermentation include whether it is likely to occur in bacteria. What are some commercially important products?

Answer 43

Lactic Acid Fermentation:

• End Products: Lactic acid
• Organisms: Common in bacteria (e.g., Lactobacillus) and animal muscle cells
• Oxygen Requirement: Anaerobic
• Energy Yield: Produces 2 ATP per glucose molecule
• Commercial Products: Yogurt, sauerkraut, kimchi
• Biochemical Pathway: Pyruvate is directly reduced to lactic acid

Alcohol Fermentation:
- End Products: Ethanol and CO2
- Organisms: Primarily occurs in yeast (e.g., Saccharomyces cerevisiae) and some bacteria
- Oxygen Requirement: Anaerobic
- Energy Yield: Produces 2 ATP per glucose molecule
- Commercial Products: Beer, wine, bread
- Biochemical Pathway: Pyruvate is converted to acetaldehyde, then reduced to ethanol

Likelihood of Occurrence in Bacteria
- Lactic Acid Fermentation:
- Common in various bacteria, especially in food production.

• Alcohol Fermentation:
  
  • Primarily occurs in yeast, but some bacteria can perform this process

Question 44

Nitrifiers;

include substrate converted, end products and importance

Answer 44

Nitrifiers
Substrate Converted: Ammonia (NH₃)

End Products: Nitrite (NO₂⁻) then nitrate (NO₃⁻)

Importance: Nitrifying bacteria (e.g., Nitrosomonas and Nitrobacter) are essential in the nitrogen cycle, converting ammonia & nitrite into forms that plants can absorb (nitrate)

Question 45

Nitrogen-fixers;

include substrate converted, end products and importance

Answer 45

Nitrogen-Fixers

Substrate Converted: Nitrogen gas (N₂)

End Products: Ammonia (NH₃)

Importance: Nitrogen-fixing bacteria (e.g., Rhizobium, Azotobacter) convert inert nitrogen gas into a usable form (ammonia) for plants, crucial for soil fertility.

Question 46

Denitrifiers;

include substrate converted, end products and importance

Answer 46

Denitrifiers

Substrate Converted: Nitrate (NO₃⁻)

End Products: Nitrogen gas (N₂)

Importance: Denitrifying bacteria (e.g., Pseudomonas) return nitrogen to the atmosphere, closing the nitrogen cycle and preventing the buildup of excess nitrate in soil and water.

Question 47

Sulfur oxidizers;

include substrate converted, end products and importance

Answer 47

Sulfur Oxidizers

Substrate Converted: Reduced sulfur compounds (e.g., hydrogen sulfide H₂S)

End Products: Sulfate (SO₄²⁻)

Importance: Sulfur-oxidizing bacteria (e.g., Thiobacillus) play a key role in the sulfur cycle by converting toxic hydrogen sulfide into sulfate, supporting plant growth.

Question 48

Sulfur reducers;

include substrate converted, end products and importance

Answer 48

Sulfur Reducers

Substrate Converted: Sulfate (SO₄²⁻)

End Products: Hydrogen sulfide (H₂S)

Importance: Sulfate-reducing bacteria (e.g., Desulfovibrio) are important in anaerobic environments, where they reduce sulfate to hydrogen sulfide, often contributing to the sulfur cycle in sediments.

Question 49

Decomposers;

include substrate converted, end products and importance

Answer 49

Decomposers

Substrate Converted: Organic matter (e.g., dead plants and animals)

End Products: Inorganic nutrients (CO₂, NH₃, H₂O)

Importance: Decomposers (e.g., fungi, bacteria) break down dead organisms, recycling nutrients back into ecosystems and supporting primary producers

Question 50

Methanogens;

include substrate converted, end products and importance

Answer 50

Methanogens

Substrate Converted: Carbon compounds (e.g., CO₂, acetate)

End Products: Methane (CH₄)

Importance: Methanogens (e.g., Methanobacterium) produce methane in anaerobic environments like swamps, sewage treatment plants, and animal guts, playing a role in carbon cycling and contributing to greenhouse gases.

Question 51

Methanotrophs;

include substrate converted, end products and importance

Answer 51

Methanotrophs

Substrate Converted: Methane (CH₄)

End Products: CO2 and H₂O

Importance: Methanotrophs (e.g., Methylococcus) metabolize methane, acting as a biological control for methane emissions and mitigating its release into the atmosphere.

Question 52

Compare & contrast photosynthesis in prokaryotes vs. eukaryotes include light-dependent

Answer 52

Photosynthesis in Prokaryotes

• Cyanobacteria: Light-dependent reactions occur in thylakoid membranes, using both Photosystems I (PSI) and II (PSII).
• Purple Bacteria: Light-dependent reactions occur in plasma membrane invaginations, using only one photosystem (similar to PSI) in a cyclic process, without water splitting or oxygen production.
• Green Sulfur Bacteria: Light-dependent reactions occur in chlorosome-bound membranes, with one photosystem resembling PSI.

Photosynthesis in Eukaryotes (Plants and Algae)

• Light-dependent reactions take place in thylakoid membranes within chloroplasts, using both PSI and PSII and involving water splitting.

Question 53

Compare & contrast photosynthesis in prokaryotes vs. eukaryotes include light-independent reactions

Answer 53

Photosynthesis in Prokaryotes

• Cyanobacteria: Light-independent reactions occur in the cytoplasm via the Calvin cycle, using ATP and NADPH to convert CO₂ into organic compounds.
• Purple Bacteria: Use alternative carbon fixation pathways, like the reverse TCA cycle, instead of the Calvin cycle.
• Green Sulfur Bacteria: Rely on the reverse TCA cycle for carbon fixation, rather than the Calvin cycle.

Photosynthesis in Eukaryotes (Plants and Algae)

• Light-independent reactions occur in the stroma of chloroplasts via the Calvin cycle, using ATP and NADPH from light-dependent reactions to fix CO₂ into sugars.

Question 54

Compare & contrast photosynthesis in prokaryotes vs. eukaryotes include locations of photosystems, source of electrons, oxygen production, & pigment composition of photosystems.

Answer 54

Photosynthesis in Prokaryotes

• Cyanobacteria:
  
  • Electron source: Uses water (H₂O), producing oxygen.
  • Oxygen production: Oxygenic, generating oxygen through water splitting in PSII.
  • Pigment composition: Contains chlorophyll a and phycobilins (such as phycocyanin and phycoerythrin) to capture light.
• Purple Bacteria:
  
  • Electron source: Uses donors like hydrogen sulfide (H₂S), organic molecules, or sulfur instead of water.
  • Oxygen production: Anoxygenic, producing no oxygen.
  • Pigment composition: Contains bacteriochlorophyll and carotenoids for light absorption.
• Green Sulfur Bacteria:
  
  • Electron source: Uses hydrogen sulfide (H₂S) or other sulfur compounds.
  • Oxygen production: Anoxygenic, with no oxygen produced.
  • Pigment composition: Contains bacteriochlorophyll (c, d, or e) and carotenoids.

Photosynthesis in Eukaryotes (Plants and Algae)

• Electron source: Uses water (H₂O), releasing oxygen.
• Oxygen production: Oxygenic, with oxygen produced from water splitting in PSII.
• Pigment composition: Contains chlorophyll a, chlorophyll b (in plants), and carotenoids as accessory pigments.

Question 55

Describe how to perform the Durham fermentation test include sugars in each

Answer 55

Procedure:

Set Up:

Prepare a test medium (usually phenol red broth) containing:
- One specific sugar (e.g., glucose, lactose, sucrose, or mannitol).
- Phenol red as a pH indicator (yellow below pH 6.8, red at neutral pH, and pink above pH 7.4).

Insert a Durham tube (a small inverted tube) inside the larger test tube to trap any gas produced during fermentation.

Inoculation:
Inoculate the medium with the test bacteria and incubate at the appropriate temperature (typically 35-37°C) for 24-48 hours.

Question 56

Discuss the purpose of Durham Fermentation as a laboratory test

Answer 56

To determine whether a microorganism can ferment specific sugars, producing acid and/or gas as metabolic byproducts.

Question 57

Describe the appearance of possible Durham Fermentation test results.

Answer 57

Possible Test Results:

1. Fermentation with acid production (positive result):

• Appearance: Medium turns yellow, indicating a drop in pH due to acid production from fermentation

2. Fermentation with acid and gas production (positive result):

• Appearance: Medium turns yellow, and the Durham tube contains a visible gas bubble.

3. No fermentation (negative result):

• Appearance: Medium remains red or turns slightly pink due to an increase in pH

Question 58

If given a photo of this test (Durham Fermentation) or an actual tube, then be able to describe the test result & what it mean with regards to bacterial metabolism.

Answer 58

Interpreting a Photo or Actual Test Tube:

Yellow color without gas: Positive for acid production, negative for gas production.

Yellow color with a gas bubble: Positive for both acid and gas production.

Red or pink color: Negative for fermentation, meaning the bacterium does not utilize the sugar for fermentation.

Question 59

Compare & contrast mixed acid fermentation vs. butanediol fermentation.

Answer 59

Comparison of Mixed Acid Fermentation vs. Butanediol Fermentation

Mixed Acid Fermentation:
- Pathway: Produces a variety of strong acids (e.g., lactic, acetic, formic, and succinic) from glucose fermentation.
- End Products: Includes strong acids, carbon dioxide (CO₂), hydrogen (H₂), and ethanol.
- pH Effect: Significantly decreases pH due to multiple acids produced.
- Bacterial Example: Escherichia coli.

Butanediol Fermentation:
- Pathway: Produces neutral end products, primarily 2,3-butanediol, along with some acids (e.g., lactic and formic).
- End Products: Mainly 2,3-butanediol, ethanol, CO₂, and some weak acids.
- pH Effect: Produces less acid, resulting in a higher pH.
- Bacterial Example: Enterobacter, Klebsiella.

Key Differences:
- Type of End Products: Mixed acid fermentation produces strong acids, while butanediol fermentation produces neutral compounds.
- Impact on pH: Mixed acid fermentation significantly lowers pH, whereas butanediol fermentation results in a higher pH.

Question 60

Which lab test(s) can be used to distinguish between the two types? (mixed acid fermentation vs. butanediol fermentation)

Describe how the test(s) is/are performed

Answer 60

Methyl Red (MR) Test:

• Purpose: detect mixed acid fermentation.

Procedure:
- Inoculate bacteria into MR-VP broth and incubate for 48 hrs.
- Add 20 drops of methyl red indicator to the MR (2/3) broth.

Results: MR
- Positive result: If broth stays red, it indicates the presence of strong acids (pH < 4.4), confirming mixed acid fermentation.

• Negative result: yellow or orange, it indicates no significant acid production, no mixed acid fermentation.

Voges-Proskauer (VP) Test:

• Purpose: detect the production of 2,3-butanediol (butanediol fermentation).

Procedure:
- Inoculate bacteria into MR-VP broth and incubate for 48 hours.
- Add Barritt’s reagents (α-naphthol and KOH) to the VP (1/3) broth. 10 drops each
- incubate for ~30 minutes.

Results: VP
- Positive result: If broth turns red or pink, it indicates the presence of acetoin; butanediol fermentation.

- Negative result: No color change or a yellow color indicates no butanediol production.

Question 61

Be able to interpret results of each if given an actual test or photo of one (Methyl Red (MR) Test & Voges-Proskauer (VP) Test)

Answer 61

Methyl Red Test:

• Red: Positive for mixed acid fermentation.
• Yellow/orange: Negative for mixed acid fermentation.

Voges-Proskauer Test:

• Red/pink: Positive for butanediol fermentation (presence of acetoin).
• No color change or yellow: Negative for butanediol fermentation.

Question 62

Do we perform any lab tests that tell us about a bacterium’s ability to metabolize proteins? If so, then name the test(s).

Answer 62

Urease Test:
Purpose: Measures the ability of a bacterium to hydrolyze urea to ammonia and carbon dioxide using the enzyme urease.
Procedure: Inoculate bacteria into a urea broth or agar that contains phenol red (pH indicator).
- Positive: The medium turns pink or red due to ammonia production, indicating urease activity. Increased pH due to ammonia
- Negative: The medium remains yellow or orange, indicating no urease activity.

Indole Test:
Purpose: Assesses whether a bacterium can produce indole from the amino acid tryptophan.
Procedure: inoculate bacteria into a tryptophan broth
-Positive: result is indicated by a pink or red color change after adding 10 drops of Kovac’s reagent.
-Negative result: remains yellow indicating no indole production

Citrate Utilization Test:
Purpose: test if bacteria can use citrate as a sole carbon source through protein metabolism pathways.
Procedure: inoculate bacteria into a citrate slant
-Positive: Color changes to blue in the medium indicates a positive result.
-Negative: turns green, bacteria is not able to utilize citrate

Question 63

Starch Hydrolysis test

Answer 63

Starch (Amylose) Hydrolysis Test

Purpose: Determines whether a bacterium produces amylase, an enzyme that breaks down starch.

Procedure:
Inoculate bacteria onto a starch agar plate and incubate.
After incubation, flood the plate with iodine (which turns blue/black in the presence of starch).

Results:
Positive: A clear zone around the bacterial growth indicates starch hydrolysis (amylase activity).

Negative: No clear zone; the area around the bacteria remains blue/black, indicating no amylase production

Question 64

Casein Hydrolysis Test (Milk Agar Test)

Answer 64

Casein Hydrolysis Test (Milk Agar Test)

Purpose: Identifies bacteria that produce caseinase, an enzyme that hydrolyzes casein (milk protein).

Procedure:
Inoculate bacteria onto a milk agar plate and incubate.

Results:
Positive: A clear zone around the bacterial growth indicates casein degradation.

Negative: No clear zone, indicating no caseinase production

Question 65

Tryptophan Hydrolysis (Indole Test)

Answer 65

Tryptophan Hydrolysis (Indole Test)

Purpose: Assesses whether a bacterium can produce indole from the amino acid tryptophan.
Procedure: inoculate bacteria into a tryptophan broth
-Positive: result is indicated by a pink or red (ring at top) color change after adding 10 drops of Kovac’s reagent.
-Negative result: remains yellow indicating no indole production

Question 66

Urea Hydrolysis (Urease Test)

Answer 66

Urease Test:

Purpose: Measures the ability of a bacterium to hydrolyze urea to ammonia and carbon dioxide using the enzyme urease.
Procedure: Inoculate bacteria into a urea broth or agar that contains phenol red (pH indicator).
- Positive: The medium turns pink or red due to ammonia production, indicating urease activity. Increased pH due to ammonia
- Negative: The medium remains yellow or orange, indicating no urease activity

Question 66

Nitrate Reduction Test

Answer 66

Nitrate Reduction Test

Purpose: Tests for the ability of bacteria to reduce nitrate (NO₃⁻) to nitrite (NO₂⁻), nitrogen gas (N₂), or ammonia (NH₃)

Procedure:
Inoculate bacteria into nitrate broth and incubate.
After incubation, add nitrate reagents A and B.

If no color change, add zinc powder to confirm.

Results:
Positive
nitrite: Broth turns red after reagents A and B.

Nitrogen gas: No color change after zinc addition.

Negative: Red color after adding zinc (no nitrate reduction occurred).

Question 67

Triple Sugar Iron (TSI) Test

Answer 67

Triple Sugar Iron (TSI) Test

Purpose: Detects carbohydrate fermentation, gas production, and hydrogen sulfide (H₂S) production.

Procedure:
Inoculate bacteria into the TSI agar slant by stabbing the butt and streaking the surface.
Incubate and observe slant and butt color changes, gas production, and H₂S production.

Results:
- Red slant/yellow butt: Ferments glucose only.
- Yellow slant/yellow butt: Ferments glucose and lactose/sucrose.
- Black precipitate: H₂S production.
- Gas production: Cracks or bubbles in the agar

Question 68

Oxidase Test

Answer 68

Oxidase Test

Purpose: Detects the presence of cytochrome c oxidase, an enzyme involved in the electron transport chain in aerobic respiration.

Procedure:
Place a few drops of oxidase reagent on a bacterial colony on q-tip

Results:
Positive: Bacteria turn dark purple/blue within 30 seconds. yes cytochrome c oxidase

Negative: No color change. no cytochrome c oxidase

Question 69

Catalase Test

Answer 69

Catalase Test

Purpose: Detects the presence of catalase, an enzyme that breaks down hydrogen peroxide (H₂O₂) into water and oxygen.

Procedure:
Place a drop of H₂O₂ on a bacterial colony or a slide containing bacteria.

Results:
Positive: Rapid production of bubbles (oxygen release).

Negative: No bubbles form. no catalase

Question 70

Citrate Utilization Test (Simmons Citrate Test)

Answer 70

Citrate Utilization Test (Simmons Citrate Test)

Purpose: Determines whether a bacterium can use citrate as its sole carbon source and ammonium ions as its sole nitrogen source.

Procedure:
Inoculate bacteria onto Simmons citrate agar slant and incubate.

Results:
Positive: The medium turns blue (alkaline pH due to citrate metabolism).

Negative: The medium remains green (no citrate metabolism).