Microbial Metabolism:

Catabolic and Anabolic Reactions

Metabolism: the sum of the chemical reactions in an organism.

Catabolism:
C6H12O2+ O2->CO2+H2O (exothermic, exergonic)

Anabolism:
energy+CO2+H2O->C6H12O6+O2 (Endothermic, Endergonic)

Provides energy and building blocks for anabolism. (makes smaller molecules)

Uses energy and building blocks to build large molecules.

Metabolic Pathway

a Sequence of enzymatically catalyzed chemical reactions in a cell

determined by enzymes

encoded by genes

Collision Theory:

Activation Energy:

Reaction Rate:

When atons, ions, and molecules collide.

disrupts electonic configurations

is the frequency of collisions with enough energy to bring about a reaction
The reaction rate can be increased by enzymes or by increasing temperature or pressure.

Enzyme Components:

Biological catalysis
- specific for a chemical ration, not used up
Apoenzyme: protein
Cofactor: nonprotein component
-coenzyme: organic cofactor
Holoenzyme: apoenzyme plus cofactor

Important Coenzymes

NAD+
NADP
FAD
Coenzyme A

Enzyme Classification

Oxidoreductase: oxidation-reduction reactions
Transferase: transfer functional groups
Hydrolase: Hydrolysis
Lyase: removal of atoms without hydrolysis
Isomerase: rearrangement of atoms
Ligase: joining of molecules;use ATP

Factors Influencing Enzyme Activity

Temperature
pH
Substrate concentration
Inhibitors

* Temperature and pH denature proteins

Enzyme Inhibitors: Feedback Inhibiton

Ribozymes

Cut and Splices RNA

Oxidation-Reduction Reactions

Oxidation: removal of elections
Reduction: gain of electrons
Redox reaction: an oxidation reaction paired with a reduction reaction

Associated with hydrogen atoms
Biological oxidations are often dehydrogenations

The Generation of ATP

ATP is generated by the phosphorylation of ADP

Substrate -Level Phosphorylation

Energy from the transfer of a high-energy PO4-, tp ADP generates ATP

Oxidative Phosphorylation

Energy released, generat ATP in the electon transport chain.

Carbohydrate Catabolism (learned in Bio 156)

The Breakdown of carbohydrates to release energy
- Glycolysis
-Krebs cycle
- Electron transport chain

Figure 5.11

A summary of Respiration

Aerobic Respiration- electron acceptor

Anaerobic Respiration: NOT O2
- yeilds less energy then aerobic respiration

Anaerobic Respiration

Electron Acceptor Products
NO3- NO2-,N2+H2O
So4- H2S+H2O
CO32- CH4+H2o

Carbohydrate Catabolism

Pathway Eukaryote Prokaryote
Glycolysis Cytoplasm Cystoplasm
Intermediate Cytoplasm Cytoplasm
Krebs cycle Mitchondrial matrix Cytoplasm
ETC Mitochondrial inner membrane
Plasma membrane

The Electron Transport Chain

electrons are passed down the chain

energy released can be used to produce ATP by chemiosmosis

Fermentation

spoilage of food

produces alcoholic beverages

large scale microbial process occurring with or without air.

-releases energy from oxidation of organic molecules
-does not require oxygen
-Does not use the Krebs cycle or ETC
- Organic molecules as the final electron acceptor

*Alcohol fermantation: produces ethanol +CO2

* Lactic acid fermantation: produces lactic acid

Chemotrophs

Use enegery from chemicals
Chemoheterotroph

Chemotrophs

Energy is used in the Calvin-Benson cycle to fix CO2

Phototrophs

Use light energy

Photoautotrophs use energy in the Calvin-Benson cycle to fix CO2

Photoheterotrophs use energy

Figure 5.27 Requirements of ATP production

Figure 5.28 A nutrional classification of Orgnaisms

* ALL ORGANISMS HAVE TO HAVE ENERGY
-CHEMICAL
-LIGHT

Metabolic Diversity among Organisms

Biological oxidation are also called ___ reactions.

dehydrogenations

At the completion of the Krebs cycle, the carbons from glucose are in ___ (name the molecules)

Carbon dioxide (co2)

At the completion of aerobic respiration,energy has been formed. The energy from the oxidation of glucose is sotred in ____?

ATP

_______ involves an inhibitor that fills the active site of an enzyme and competes with the normal substrate for the active site.

Competive inhibition

For every NADH that is oxidized via the electron transport chain,___ ATP are formed.

3

All of the following pairs are correctly matched Except

Oxidation: reaction where are gained

Competitive inhibition of enzyme action evolves

Competition with the substrate for binding at the active site.

During glycolysis, elctrons from the oxidation of glucose are transferred to

NAD+

Streptococcus (In italics) bacteria lack an electron transport chain. How many molecules of ATP can a Streptococcus (In italics) cell net from one molecule of glucose?

2

Unlike eukaryotes, in prokaryotes chemiosmosis

Occurs at the plasma membrane an not the mitochondria.

The chemical reactions involved in synthesizing proteins and cell wall peptidoglycan are examples of _____ reactions.

anabolic

Fopre a cell wall, which of the following compounds has the greatest amount of energy per molecule?

Acetyl CoA pyruvate

The complete oxidation of glucose typically involves which three stages?

Glycolysis,Krebs cycle, and the elctron transport chain.

Which of the following mechanisms does not generate ATP using an electron transport train?

Substrate-level phosphorylation

Which of the following are products of the light- dependent reactions, on which the light dependent reactions are dependent.

ATP and NADPH

Enzymes work most effectively at their optimal temperature and pH.

True

Feedback inhibition generally acts on the last enzyme in an anabolic pathway

False

Glycoysis produces ATP through substrate phosphorylation

True

The synthesis of sugars by using carbon atoms from Co2 gas is called carbon fixation.

True

Lipids, proteins, and sugars all may serve as substrates of glycolysis.

False

Which of the following is true of catabolism?

Catabolism reactions are exergonic; they break down complex organic molecules into simpler ones.

Which of the following statements is true about enzymes?

Each enzyme has a characteristic three- dimensional shape.

The chemical reactions involved in synthesizing proteins and cell wall peptidoglycan are examples of ____ reactions.

anabolic

Which of the following statements is accurate concerning glucose metabolism?

Pyruvic acid, the product of glycolysis, is the starting block for both Krebs cycle and fermentation.

The complete oxidation (catabolism) of glucose typically involves three stages. The greatest amount of ATP is produced in which stage?

The elctron transport chain

Which of the following molecules carry electrons during various stages of glucose catabolism?

NADH & FADH2

Which of the following four stages of glucose oxidation requires molecular oxygen?

Oxidative phosphorylation

Which o fthe following statements accurately describes the difference between aerobic & anaerobic respiration?

Aerobic respiration use oxygen as the final electron acceptor & anaerobic respiration uses either an inorganic molecule, such as nitrate ions or sulfate ions, or an organic molecule, such as an acid or alcohol.

Carbon fixation occurs during______.

Photosynthesis

Fermentation differs from anaerobic respiration in all of the following ways EXCEPT that fermentation does NOT ___.

use an electron transport chain.

When fermentation test are used to help identify bacteria, which of the following end-products is typically detected bya color or change?

Acid

Which of the following are products of light -dependaent ( in italics) photosynthetic reactions & are necessary for the light- independent (in italics) photosynthetic reactions occur?

ATP& NADH

During the light-independent reactions of photosynthesis, organisms ____.

Synthesize sugars

Nitrobacter (italics) bacteria use carbon dioxide for their source and Nitrate ions as an energy source. This organism is a ____.

Chemoautotroph

Which group of organisms has members representing each of the following nutrional classification: chemoheterotrophs, chemoautogrophs, photoheterotrophs,&photoautotrophs?

Bacteria

Which of the following molecules traps energy released during oxidation reduction reactions?

ATP

Some amino acids are synthesized by adding an amine group to pyruvic acid or to one of the Krebs cycle intermediates. This process is known as ____.

Amination

You inocculate a bacterial culture into a tube containing glucose & peptides. The pH indicator shows that the pH decreased after 24 hours & then increased at 48 hours. Ehat has caused the increase in pH?

Deamination

Which of the following terms refers to pathways that can function both in anabolism & catabolism?

Amphilbolic pathways

Like glucose, amono acids are catabolized for energy, but these must be converted to a form where they can enter the Krebs cycle for oxidation. All of the following reactions occur in the catabolism or amino acids EXCEPT____.

carbon fixation

Microbial Growth

Increase in number of cells, not cell size

The Requirements for Growth

*Physical Requirements
- Temperature
-pH
-Osmotic pressure
*Chemical Requirements
-Carbon
- Nitrogen, Sulfure, and Phosphorous
- Trace elements
-Oxygen
-Organic Growth factor

Physical Requirements

* Temperature
-Minimum
-Optimum
-Maximum

Figure 6.1 Typical Growth rates of different types of microorganisms

Psychrotrophs

Grow between 0 degrees Celcius and 20-30 degrees C
-cause food spoilage

pH

Most bacteria pH 6.5 and 7.5
Molds and yeasts pH 5 and 6
Acidophils grows in acidic environments

Osmotic Pressure

Hypertonic environments, increase in salt or surgar, plasmolysis

Extreme or obligate halophiles require osmotic pressure ( or they die)

Facultative halophiles tolerate

Chemical Requirements
* Carbon

-structural organic (has to have carbon in it) molecules, energy source
-Chemoheterotrophs use organic carbon sources
- Autotrophs use CO2

* Nitrogen

-In amino acids and proteins
-decompose proteins
-use NH4+or No3-
- few use N2 in nitrogen fixation

*Sulfur

* Phosphorus

In amino acids, Thiamine, and biotin
-decompose proteins
- use SO42- or H2S

* DNA, RNA, ATP, and membranes
* PO43-

Trace elements

Inorganic elements
* enzymes cofactors

Organic Growth Factors

-Organic Compounds obtained from the environment
- Vitamins, amino acids, purines and pyrimidines

Biofilms

Microbial communiites
-Form slime or hydrogels
- attracted by chemicals quorum sensing

Advantages of Biofilms

share nutrients
sheltered

Biofilms

indwelling catheters
low to cause infection
after exposure, patients developed infections

Agar

*Complex polysaccharide
-soildifying agent
-plates, slants, and deeps
* not metabolized by microbes
-liquefies at 100 degrees C
- Solidifies at -40 degree C

Culture Media

Culture medium: growth
Sterile: no living microbes
Inoculum: introduction of microbes
Culture: microbes growing
Chemically defined media: exact composition is known
Complex media: extracts and digests of yeasts, meat, or plants
- nutrient broth
- nutrient agar

Anaerobic Culture Methods
* Reducing Media

Contain chemicals (thioglycolate or oxyrase) that combine O2.
Heated to drive off O2.

Capnophiles

Require high CO2 conditions
CO2 packet
Candle jar

Biosafety levels

BSL-1: No special precautions
BSL-2: lab coat,gloves,eye protection
BSL-3: biosafety cabinets to prevent airborne transmission
BSL-4: sealed, negative pressure
*Exhaust air is filtered twice

Differential Media

Distinguish colonies of different microbes

Selective Media

Suppress unwanted microbes, encourages desired microbes

Enrichment Culture

Encourages growth of desired microbes

Obtaining Pure Cultures

Contains only one species or strain
COLONY a population of cells arising from a single cell or spore or from a group of attached cells
Colomy = Colony-forming unit
Streak plate method: isolate pure cultures

Preserving Bacterial Culutres

Deep- Freezing -50C to -95C
LYophilization (freeze-drying): vacuum

Reproduction in Prokaryotes

Binary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments

Understanding the Bacterial Growth Curve

1.Lag Phase: Intense activity preparing for population growth but no increasein population

2. Log phase:logarithmic or exponential increase in population

3. Stationary phase: Period of equilibrium; microbrial deaths balance production or new cells.

4. Death phase: Population is decreasing at a logarithmic rate

measuring Microbial Growth

Direct Methods
* Plate counts
* Filtration
* MPN
* Direct microscopic count

Indirect Methods
* Turbidity
* Metabolic activity
* Dry weight

Terminology of Microbial Control

Sepsis: contamination
Asepsis: absence of significant contamination
* Aseptic surgery- prevent microbial contamination of wounds.

Cont. Terminology of Microbial Control

Sterilization: removing all microbial life
Commerical sterilization: killing C. botulinum endospores
Disinfection: removing pathogens
Antisepsis: removing pathogens from living tissue

Cont. Terminology of Microbial Control

Degerming: removing micobes from a limited area
Sanitization: lowering microbial counts (eating utensils)
Biocide/germicide: killing microbes
Bacteriostasis: inhibiting not killing microbes

Effectiveness of treatment

Depends on:
-number of microbes
-environment
-Time of exposure
-Microbial characteristics

Actions of Microbial Control Agents

Alteration of membrane permeability
Damamge to proteins
Damamge to nucleic acids

Heat

Thermal death point (TDP): lowest temperature, all cells in a culture are killed in 10 min

Thermal death time (TDT): time during all cells in a culture are killed

Decimal Reduction Time (DRT)

Minutes to kill 90% of a population

Moist Heat Sterilization

denatures proteins
Autoclaves steam under pressure

Pasteuization

Reduces spoilage
Equivalent treatments
- 63 C for 30 min
- High-temperature short-time: 72 C for 15 min
- Ultra-high-temperature: 140 C for <1 sec
- Thermoduric organisms survive

Dry Heat Sterilization

* Kills by oxidation
- Dry heat
- flaming
- incineration
- Hot-air sterilization

Filtration

Hepa remmoves microbes
Mebrane filtration removes microbes

Physical Methods of Microbial Control

Low temperature : Inhibits growth
-Refrigeration
- Deep-Freezing
- Lyophilization
High Pressure: denautres proteins
Desiccation: prevent metabolism
Osmotic pressure: causes plasmolysis

Radiation

Ionizing radiation: x-rays, gamma rays, electrom beams

Nonionizing radiation: UV, 260nm

Microwaves: kill by heat, not especially antimicrobial

Principle of Effective Disinfection

Concentration of disinfectant
Organic matter
pH
time

Use-Dilution Test

Metal rigs dipped in test bacteria are dried

placed in disinfectant for 10 min at 20 C

transferred to culture media to determine bacteria survued treatment