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AU / Engineering / BIOL / How do microbes uptake nutrients?

How do microbes uptake nutrients?

How do microbes uptake nutrients?


School: Auburn University
Department: Engineering
Term: Spring 2019
Cost: 50
Name: Microbiology Exam 2 Study Guide
Description: Week 5-7 Material
Uploaded: 02/23/2019
24 Pages 33 Views 5 Unlocks

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How microbes uptake nutrients?

Microbiology Exam 2 Study Guide  

I. How Microbes Uptake Nutrients  

A. Essential nutrients are those that must be supplied  

 1. Macronutrients- required in large quantities  

a) C, O, H, P, N, S 

b) Ions necessary for protein function: Mg 2+, Ca 2+, Fe 2+, K+  2. Micronutrients- required in small quantities 

a) Trace elements necessary for enzyme function

b) Co, Cu, Mn, Zn, Mo, Ni

B. How microbes build biomass

1. All of earth’s life-forms are based on carbon, which they acquire in  different ways

How microbes build biomass?

2. Heterotrophs use preformed organic molecules (release CO2)  3. Autotrophs fix CO2 and assemble into organ molecules (mainly  sugars)

 a) Phototrophs- use light energy 

 b) Lithotrophs- energy from the oxidation of minerals 

C. The Carbon Cycle

1. Heterotrophy- turns polysaccharides, lipids, peptides, and lignin into CO2 and H2O If you want to learn more check out Epistemology is the study of what?
We also discuss several other topics like What is the most dramatic force shaping the destiny of markets and marketing?

2. Autotrophy uses light absorption and mineral oxidation into energy  to convert CO2 and H2O into glucose  

D. The Nitrogen Cycle

1. Nitroegnsase fixes atmospheric N2 to ammonia  

2. Nitrifies oxidize NH4+ to generate energy

3. Denitrifiers use oxidized forms as alternative electron acceptors  E. Nutrient uptake

how microbes are cultured?

1. Membranes are designed to separate what is outside the cell from  what is inside  

2. Transport across a membrane happens in two ways:

a) Passive transport doesn’t require energyDon't forget about the age old question of Where was the tango born?

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 (1) Facilitated diffusion- helps solutes move across a membrane  from a region of high concentration to one of lower  


(a) It does not use energy

(b) ex. Aquaporin family transports water and small polar  

molecules such as glycerol  

(2) Simple diffusion

b) Active transport requires energy  

 (1) Coupled transport 

(a) Driver- an atom or molecule that causes the transportation to occur  

(b) Symport- two molecules travel in the same direction  

(c) Antiport- the actively transported molecule moves in the  direction opposite to the driving ion

 (2) ABC transporters - ATP-binding cassette superfamily  

(a) They are found in all three domains of life  

(b) Two main types

i) Uptake ABC transporters- transport nutrients

ii) Efflux ABC transporters- multidrug efflux pumps; multi

drug resistance  

(c) Uptake ABC transporters Don't forget about the age old question of Define biological evolution and some mechanisms that drive it.

i) Solute binds to binding protein, and the complex then  

binds to the membrane transporter  

ii) The ATPase activity of one component powers the  

opening of the channel  

iii) Movement of the solute into the cell  

II. How microbes are cultured

A. Microbes in nature exist in complex, multi species communities, but for detailed studies they must be grown separately in pure culture  1. Only 0.1% of all the microorganisms are cultured  If you want to learn more check out Where can the proteins go after they pass through the golgi?

2. The vast majority has yet to be tamed

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B. Bacteria are grown in culture media; two main types: 

1. Liquid or broth- useful for studying the growth characteristics of  pure culture  

2. Solid (gelled with Agar)- useful to separate mixed cultures  C. Types of Media: can be more than one of these (ex. Sheep blood agar  is both enriched and differential)  

 1. Complex media are nutrient rich but poorly defined; allows most  types of microbes to grow

 2. Synthetic media are precisely defined

 3. Enriched media are complex media to which specific blood  components are added (fastidious= requiring blood)

 4. Selective media favor growth of one organism over another based  on what specific nutrients are put in the media; ex. Add antibodies  to kill one bacteria but not the other (such as penicillin)  Don't forget about the age old question of Does the upper extremities are not a part of the axial skeleton?

 5. Differential media exploit differences between two species that  grow equally well  

a) E. coli vs. Salmonella  

b) MacConkey medium- differs between lactose fermenting and  non-fermenting  

(1) Lactose  

(2) Peptones

(3) Neutral red (pH) indicator (red=acid)

D. Isolating Pure Colonies- two main techniques: can be used to  differentiate between multiple types of bacteria based on colony size,  color, or shape

 1. Dilution streaking- dragging a loop across the surface of an agar  plate

 2. Spread Plate by Serial Dilution  

a) Serial dilutions are performed on a liquid culture (9 parts saline  and 1 part concentrated broth)

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(1) The next tube uses the diluted concentration created from the first tube, and so on and so forth

(2) The resulting concentrations based off the original broth:  10^-1 (tube 1), 10^-2 (tube 2), 10^-3 (tube 3), etc. (usually  stops at 10^-6)

 (3) Diluted concentration= 10^-5, then Dilution Factor (DF)=  10^5 

 (4) CFU/ml => (number of colonies x DF)/(volume transferred to  plate) 

b) A small amount of each dilution is then plated

c) Plate with isolated colonies is used (goal plate has 30-300  colonies)  

III. How microbes are counted  

A. There are may reasons why it is important to know the number of  organisms in a sample

1. There are multiple ways to count bacteria

2. Each of the available techniques measure a different physical or  biochemical aspect of growth

 B. Direct microscopic count- counted on a special microscope slide called  a counting chamber; calculate number of cells per given cubic space  (ex. 16 cells in .2 mm cubed ==> 80 cells per cubic mm)

 C. Fluoresce-Activated Cell Sorter (FACS) 

1. Fluorescent cells are passed through a small opening and then past  a laser

2. Detectors measure light scatter

a) Measure particle size  

b) Measure particle shape

D. Other counting techniques

 1. Viable cell count- count replicating and colony forming cells (pour  plate method)

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 2. Optical density (spectrophotometer)- the fastest way to measure  cell density

a) Measure the light passed through broth

b) Then compare that to a standard tube with the exact number  cells in it  

 3. Pour Plate Method- best method; counts live cells

a) Tenfold serial dilutions are performed on a liquid culture b) A small amount of each dilution is then plated

c) A plate with 30-300 colony forming units (CFU) is counted (not  called single cells because there may be more than one cell  forming that colony)

 d) Steps: 

(1) Calculate total dilution at each tube  

(2) Find the plate that contains between 30 and 300 colonies  (usually 5th or 6th plate, all others are TNTC- too numerous to count)

(3) Determine the total volume transferred to the plate that  contains 30-300 colonies  

(a) The resulting concentrations based off the original broth:  10^-1 (tube 1), 10^-2 (tube 2), 10^-3 (tube 3), etc.  

(usually stops at 10^-6)

 (b) Diluted concentration= 10^-5, then Dilution Factor (DF)=  10^5 

 (c) CFU/ml => (number of colonies x DF)/(volume transferred  to plate) 

e) However, you must assume each colony formed from a single  cell  

f) *See notes about serial dilution in isolating colonies section IV. Chapter summary

A. Microbes require certain essential macronutrients and micronutrients  to grow

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B. Microbes are classified on the basis of their carbon and energy  acquisition

C. Transport systems can be divided into two main types:

1. Passive transport does not require energy  

a) Simple diffusion

b) Facilitated diffusion

2. Active transport requires energy

a) Coupled transport  

b) ABC transporters  

D. Bacteria can be cultured on solid or liquid media  

E. Microorganisms in future may be counted directly or indirectly  V. The Microbial Growth Cycle  

A. Most bacteria divide by binary fission, where one parent cell splits into  two equal daughter cells

1. However, some divide asymmetrically  

2. Hyphomicrobium divides by budding  

 B. Exponential Growth- the growth rate, or rate of increase in cell  numbers or biomes, is proportional to the population size at a given  time  

1. Such a growth rate is called “exponential” because it generates an  exponential curve  

2. If a cell divides by binary fission, the number of cells is proportional  to 2^n (n= number of generations)

3. Some cyanobacteria divide by multiple fission  

C. Generation Time

1. Generation time- the time it takes for a population to double; G=t/n  (t= time interval in hours or minutes

 2. For cells undergoing binary fission- F= (N)(2^n) 

a) F= the final number of cells  

b) N= the original cell number

c) n= the number of generations

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d) To find n => n=log2(F/N) 

3. Problem: A virus such as influenza might produce 800 progeny virus particles from one host cell infected by one virus. How would you  mathematically represent the exponential growth of the virus?

4. Problem: A bacteria has a generation time of 20 minutes. Starting  with one cell in log phase, how many minutes does it take to  produce about 1000 cells?

D. Stages of Growth in a Batch Culture

1. Exponential growth never last indefinitely  

2. The simplest way to mode the effects of a changing environment is  to culture bacteria in a batch culture  

3. Batch culture- a liquid medium within a closed system  

4. The changing conditions in this system greatly affect bacterial  physiology and growth  

5. This illustrates the remarkable ability of bacteria to adapt to their  environment  

E. Growth Curve Stages 

 1. Lag Phase- bacteria prepare to multiply by gathering nutrients   2. Log Phase- growth approximates an drastic exponential curve  upwards

 3. Stationary Phase- Cell stops growing and shut down growth  machinery while turning on stress responses to help retain viability   4. Death Phase- cells die in a “half-life” similar to radioactive decay, a  negative exponential curve; cellular environment is waste filled  F. Continuos Cultures  

1. In a continuous culture, all cells in a population achieve a steady  state, which allows detailed study of bacterial physiology  

2. The chemostat ensures logarithmic growth by constantly adding  and removing equal amounts of culture media  

3. Note that the human gastrointestinal tract is engineered much like  a chemostat

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 VI. Biofilm 

A. In nature, many bacteria form specialized, surface attached  communities called biofilms

1. These can be constructed by one or more species, and can form a  range of organic or inorganic surface  

2. The biofilm that forms on teeth is called plaque  

B. Biofilm Development- bacterial biofilms form when nutrients are  plentiful  

1. Biofilms in nature can take many different forms and serve different functions for different species  

2. The formation of biofilms can be cued by different environment  signals in different species  

VII.Cell Differentiation  

A. Bacteria faced with environmental stress undergo complex molecular  reprogramming that includes changes in cell structure

B. Examples: neither are very common among microbes  

1. Endospores of Gram positive bacteria

2. Fruiting bodies of Myxococcus xanthus  

 C. Endospores  

1. Clostridium and Bacillus species can produce dormant spores that  are heat resistant

2. Spore dormancy is due to the tough, protective outer layer  3. Starvation initiates an elaborate 8-hour genetic program that  involves an asymmetrical cell division process that produces a  forespore and ultimately an endospore

4. Sporulation can be divided into discrete stages based on  morphological appearance  

5. Endospore Stages:

a) Septum forms near one pole. DNA replicates and extends into an axial filament

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b) Septum separates forespore from mother cell. DNA pumped  through septum until each compartment gets a chromosome. c) Mother cell engulfs forespore, surrounding it with a second  membrane.

d) Chromosomes of mother cell disintegrates.

e) Forespore develops a cortex layer of peptidoglycan between  original forespore membrane and the membrane from the  mother cell. Coat proteins deposited on outer membrane.  

f) Dipicolinic acid is synthesized, and calcium is incorporated into  the spore coat.

g) Mother cell releases spore.

 A. Fruiting Bodies 

1. Myxococcus xanthus uses gliding motility  

2. Starvation triggers the aggregation of 100,000 cells, which form a  fruiting body

I. Chapter Summary

A. The growth cycle of organisms grown in liquid batch culture consists of four phases

1. Lag

2. Log(arithmic)

3. Stationary  

4. Death

B. Biofilms are complex, multicellular, surface-attached microbial  communities  

C. Many bacteria can undergo cell differentiation (ex. Endospores and  fruiting bodies)

VIII. Environment Influence on Growth

 A. The “normal” growth conditions are: 

1. Elevation: Sea level

2. Temp: 20C to 40C

3. pH: neutral (around 7)

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4. Other: 0.9% salt, and ample nutrients 

B. Any ecological niche outside this window is called “extreme,” and  organisms inhabiting them are called extremophiles 

C. Temperature  

1. Adaptation to Temperatures

a) Psychrophiles: 0C-20C: cold loving microorganisms; membrane  filled with unsaturated fatty acids to maintain a flexible  


b) Mesophiles: 15C-45C: the temperature humans live with, so  many of these will use humans

c) Thermophiles: 40C-80C

d) Hyperthermophiles: 65C-121C: in order to prevent liquidation,  the fatty acids will be saturated  

2. Microbial Response to Temperature  

a) The thermophile Thermus aquaticus was the first source of high temperature DNA polymerase used for PCR amplification (DNA  polymerase is active at 100C)

D. Pressure  

1. Barosensitive- organisms die as pressure increases

2. Barophiles- require high pressure to grow, but die at higher pressure 3. Barotolerant- grow up to a certain pressure, but die at higher  pressures

4. High pressures occur closer to the Earth’s core (ex. Mariana Trench)  and low pressures occur farther from the Earth’s core (ex. Mount  Everest)  

E. pH- affects the activity of enzymes; measures acidity in hydrogen ion  concentration

1. Majority of enzymes operate best between pH 5 and 8.5 2. Neutralophiles- grow at pH 5 to 8, ex. Most human pathogens 3. Acidophiles- grow at pH 0 to 5, ex. Are often chemoautotrophs 4. Alkaliphiles- grow at pH 9 to 11, ex. Typically found in soda lakes

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F. Oxygen and other Electron Acceptors 

1. Many microorganisms can grow in the presence of O2

2. Some even use the oxygen as a terminal electron acceptor (TEA) in  the electron transport chain (aerobic respiration)

3. Benefits and Risks  

a) Oxygen is a benefit to aerobes, organisms that can use it to  extract energy from nutrients  

(1) Strict aerobes- only grow in oxygen

(2) Microaerophiles- grow only at low O2 levels  

b) Oxygen is toxic to all cells that do not have enzymes capable  efficiently destroying the Reactive Oxygen Species (ROS) ex.  Anaerobes  

(1) Strict anaerobes- die in the least bit of oxygen

(2) Areotoleraat anaerobes- grow in oxygen while retaining a  fermentation-based metabolism

(3) Facultative anaerobes- can live with or without oxygen (they  possess both the ability for fermentative metabolism and  


 4. Culturing Anaerobes in the Lab: three techniques 

a) Special reducing agents (thioglycolate) or enzyme systems  (oxyrase) can be added to ordinary liquid media

b) An anaerobe jar- O2 is removed by a reaction catalyzed by  palladium (O2 + 2H2 —> 2H2O)

c) An anaerobic chamber with glove ports- O2 is removed by  vacuum and replaced with N2 and CO2

IX. Controls of Microbial Growth

A. A variety of terms are used to describe antimicrobial control measures: 1. Sterilization- killing of all living organisms  

2. Disinfection- killing or removal of pathogens from inanimate objects 3. Antiseptics- killing or removal of pathogens from the surface of  living tissues

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4. Sanitation- reducing the microbial population to safe levels B. Physical agents that kill microbes 

1. Temperature  

 a) High temperature  

(1) Moist heat is more effective than dry heat

(2) Boiling water kills most cells

(3) Killing spores and thermophiles usually requires a  

combination of high pressure and temperature  

(4) Steam autoclave (pressure cooker)- 121C at 15 psi for 20  minutes; only hyperthermophiles can survive in it  

b) Pasteurization- different time and temperature combinations can be used:

(1) LTLT (low temp and long time)- 63C for 30 min

(2) HTST (high temp and short time)- 72C for 15 seconds

(3) UHT (ultra-high temp)- 134C for 2 seconds (mainly used to  sterilize milk)

 c) Cold 

(1) Low temperature slow growth and preserve strains

(2) Refrigeration temps (4C-8C) are used for food preservation (3) For long term storage of cultures:

(a) Placing solutions in glycerol at -70C

(b) Lyophilization = freeze-drying

2. Filtration- micropore filters with pore sizes of .2 micrometers can  removed microbial cells, but not viruses, from solutions  

a) Laminar flow biological safety cabinets force air through filters,  which remove >99.9% of airborne particulate material .2  

micrometers in size or larger  

 3. Irradiation 

a) Ultraviolet light (UV)

(1) Has poor penetration power

(2) Used only for surface sterilization

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b) Gamma rays, electron beams, and X-rays 

(1) Have high penetrating power

(2) Used to irradiate foods and other heat-sensitive items

(3) Astronaut food is sterilized with gamma rays

c) Can microwave kill bacteria?

 C. Chemical Agents: Disinfectants and Antiseptics  

1. These include:

a) Ethanol (70% is used because if it is too concentrated it will  penetrate the cell and it will evaporate instantly)

b) Iodine  

c) Chlorine  

d) Ethylene oxide- a gas sterilant for heat and moisture sensitive  materials

2. These damage proteins, lipids, and/or DNA- are used to reduce or  eliminate microbial content from objects

D. Antibiotics- compounds synthesized by one microbe that kill or inhibit  the growth of other microbial species  

1. Penicillin mimics peptidoglycan of the bacterial cell wall; it then  prevents cell wall formation

2. Other antibiotics target:

a) Protein synthesis  

b) DNA replication

c) Cell membranes  

 E. Biological Agents: Biocontrol- the use of one microbe to control the  growth of another

1. Probiotics contain certain microbes that, when ingested, aim to  restore balance to intestinal flora; can be harvested from fecal  matter- can be prescribed fecal tablets  

2. Phage therapy aims to treat infectious diseases with a virus  targeted to the pathogen  

X. Chapter Review

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A. Microbes inherit various niches based on their tolerance to the physical and chemical conditions

B. Microbes are classified by growth temp: psychrophiles, mesophiles,  thermophiles

C. Basophils can grow at very high pressure  

D. Halophiles require high salt concentrations  

E. Halophytes require high salt concentrations  

F. Microbes are classified by pH range: acidophiles, neutralophiles, and  alkaliphiles  

G. Microbes are classified by their O2 requirements: aerobes, facultative,  microaerophiles, and anaerobes  

H. Cells treated with antimicrobials die at a logarithmic rate I. Physical agents used to control microbes: autoclaving, pasteurization,  refrigeration, filtration, and irradiation  

J. Chemical agents used to control microbes: antiseptics and  disinfectants  

K. Antibiotics selectively control microbial growth  

L. Biological control of microbes: probiotics and phage therapy  XI. Introduction Catabolism and Energy Production  

A. There are two main energy sources:

1. Light

a) Phototrophs convert light into ATP using photography, ex.  Rhodobacter capsulatus

2. Chemicals: chemotrophy

a) Chemolithotrophs concert inorganic chemicals into ATP, ex.  Thiobacillus thiooxidans  

b) Chemoorganotrophs convert organic chemicals into ATP, ex. E.  coli  

B. All living cells need energy to move and grow

1. Catabolism- breakdown complex molecules into simpler ones 2. Anabolism- reactions that build cells

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C. Catabolism provides energy for anabolism, usually in the form of ATP D. Metabolism is comprised of anabolism and catabolism  

XII. Electron Carriers and Electron Transfer  

A. Many of the cell’s energy transfer reactions involve energy carriers  1. Molecules that gain or release small amounts of energy in reversible reactions


B. Energy carriers can also transfer electrons  

C. NADH- electron donor

1. Nicotinamide adenine dinucleotide (NADH) carries two or three  times as much energy as ATP

2. NADH is the reduced form

3. Overall, reduction of NAD+ accepts two hydrogen atoms and two  electron to make NADH: (NAD+) + (2H+) + (2e) => NADH + (H+) D. NAD+- electron acceptor  

1. NAD+ is the oxidized form

E. Flavin adenine dinucleotide (FAD) is another coenzyme that can  transfer electrons  

1. FADH2 is the reduced form  

2. FAD is the oxidized form

3. Unlike NAD+, FAD is reduced by 2e AND 2 protons

 F. ATP 

1. Adenosine triphosphate (ATP) contains a base, a sugar, and three  phosphates  

2. ADP + inorganic phosphate => ATP

3. ATP contains three phosphate molecules that yield energy upon  hydrolysis  

4. ATP transfer energy in three different ways:

a) Hydrolysis-releasing phosphate (Pi)

b) Hydrolysis-releasing pyrophosphate (PPi)

c) Phosphorylation of an organic molecule

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 5. Substrate-level phosphorylation  

a) Transfer of phosphate from high-energy molecule to ADP  b) Requires a kinase enzyme  

XIII. Catabolism- the microbial buffet  

A. Microbes catalyze many different kinds of substrates  

1. Polysaccharides are broken down to pyruvate: glycolysis  2. Pyruvate are fermented or further catabolized to CO2 and H2O via  the TCA cycle  

3. Lipids and amino kids are catabolized to glycerol and acetate, as  well as other metabolic intermediates

 B. Glucose breakdown  

1. Bacteria and archaea use three main routes to catabolize glucose  a) Glycolysis, or Embden-Meyerhof-Parnas (EMP) pathway  b) Entner-Doudoroff (ED) pathway

c) Pentose phosphate pathway (PPP), also known as the pentose  phosphate shunt  

2. Glycolysis 

a) It is central for animals and plans, as well as may bacteria and  archaea  

b) It occurs in the cytoplasm of a cell

c) It functions in the presence or absence of O2

d) It involves ten different distinct reactions that are divided into  two stages:

(1) Energy Investment  

(a) Glucose is “activated” by 2 phosphorylations  

i) Two ATPs are expended  

(b) Fructose-1,6-biphosphate is cleaved into two 3-carbon  


i) DHAP- dihydroxyacetone phospate  

ii) G3P- gyceraldehyde-3-phosphate

(2) Energy Yield

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(a) Each (two) glyceraldehyde-3-phosphate molecule is  

ultimately converted to pyruvate  

(b) Redox reactions produce two molecules of NADH

(c) Four ATP molecules are produced by substate-level  


i) Net ATP= (total — invested)

ii) 4-2= 2 ATP

 e) Summary  

(1) Primary pathway to convert one glucose to two pyruvate  (2) Pathway generates:

(a) 2 pyruvate

(b) 2 net ATP

i) 2 ATP expelled to break glucose  

ii) 4 ATP harvested  

(c) 2 NADH (will generate more ATP later)

XIV. Fermentation 

 A. Microbes use pyruvate to recycle NADH to NAD+ and to produce acid  and/or alcohol 

 B. Fermentation is the completion of catabolism without the electron  transport system and a terminal electron acceptor  

1. NADH donate H+ and electrons back to pyruvate  

2. NADH will be converted to NAD+

 C. Fermentation is not efficient (which is why it results in many  byproducts); therefore it consumes many sugary molecules  1. Formic acid

2. CO2 and H2

3. Acetic Acid

4. Ethanol

5. Lactic acid

6. Succinate acid

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 D. Most fermentations do not generate ATP beyond that produced by  glycolysis  

1. Microbes compensate by consuming large quantities of substrate ad by…

2. Excreting large quantities of byproducts  

E. Fermentation pathways: 

1. Homolactic fermentation- produces two molecules of lactic acid  2. Ethanolic fermentation- produces two molecules of ethanol and two  CO2

3. Heterolactic fermentation- produces one molecule of lactic acid, one ethanol, and one CO2

4. Mixed-acid fermentation- produces acetate, formate, lactate, and  succinate, as well as ethanol, H2, and CO2

F. Another important application of fermentation lies in diagnostic  microbiology  

1. To quickly identify the microbe causing a disease and prescribe  effective antibiotic, hospitals use rapid and inexpensive biochemical tests

2. ex. Phenol red test

3. Methyl Red (MR) and Voges-Proskauer (VP)

a) It is a simple broth that contains peptone, buffers, and glucose b) Methyl red differs from phenol red in that it is yellow at pH6.2  and above and at pH 4.4 and below

(1) MR turns red if the organisms uses the mixed acid  

fermentation pathway to make fatty acids  

(2) VP tests for organisms that use butylene glycol pathway and  produce acetoin

(a) Positive- deep red

(b) Negative- copper color

G. Fermentation is a partial breakdown of organic food without the use of  an electron transport system

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II. Introduction to Catabolism after Glycolysis: 2 pyruvate are ready to be  used  

A. The TCA cycle is also known as the Krebs cycle, or the citric acid cycle  1. Occurs in the cytoplasm for prokaryotes  

 2. Occurs in the mitochondria for eukaryotes  

3. (Both prokaryotes and eukaryotes have glycolysis occur in the  cytoplasm)

 B. Glucose catabolism connects with the TCA cycle through pyruvate  breakdown of acetly-CoA and CO2 

 1. One glucose molecule produces two pyruvate 

2. Acetyl-CoA enters the TCA cycle by condensing with the 4-C  oxaloacetate to form citrate  

3. Acetyl-CoA is composed of pyruvate, fatty acids, vanillin, and amino acids 

 XV. Pyruvate Breakdown to Acetyl-CoA 

A. Conversion of pyruvate to acetyl-CoA is catalyzed by a very large  multisubunit enzyme called the pyruvate dehydrogenase complex  (PDC)

B. Pyruvate + (NAD+) + CoA => Acetyl-CoA + CO2 + NADH + (H+) XVI. TCA Cycle 

A. Steps:

1. Hydrolysis of acetyl-CoA provides energy for condensing the acetyl  group with oxaloacetate  

2. Oxidative decarboxylation of isocitrate; 2-Oxoglutarate  intermediate is produced at this point

3. Oxidative decarboxylation; CoA is incorporated into succinyl-CoA 4. 2H+ and 2e are transferred to form FADH2; succinate is converted  to fumarate  

5. Water is incorporated, 2H+ and 2e are transferred to NADH and H+, and malate is converted to oxaloacetate (intermediate produced) B. For each pyruvate oxidized:

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a) 3 CO2 (one produced by Acetyl-CoA composition and two  through TCA) are produced by decarboxylation

b) 4 NADH (three in TCA and one in Acetyl-CoA composition) and 1  FADH2 are produced  

c) 1 ATP is produced by substrate-level phosphorylation  

(1) Some cells make GTP instead

(2) However, GTP and ATP are equivalent in stored energy  

 XVII. Glyoxylate bypass 

A. When glucose is absent, cells can catabolize acetate or fatty acids  using a modified TCA cycle called the glyoxylate bypass

1. Consists of two enzymes that divert isocitrate to glyoxylate and  incorporate a second acetyl-CoA to form malate (skips step 2 and 3  of TCA)

2. The glyoxylate bypass cuts loss of CO2

3. Helps P. aeruginosa and M. tuberculosis persist for long periods  within lung macrophages  

B. This will occur if there is not enough carbon to go around  XVIII. Electron Transport Systems (ETS) 

A. Intro

 1. Each glucose consumed: 10 NADH and 2 FADH2 produced  a) Two NADH in Glycolysis

b) Two NADH in Transition Step (Pyruvate to Acetyl-CoA)

c) Six NADH and two FADH2 in TCA

 2. BIG Picture: 

a) Microbes transfer energy by moving electrons

(1) Electrons move from reduced molecules to energy carriers… (2) From electron carriers to membrane protein carriers…  

(3) Finally from from membrane protein careers to oxygen to  oxidized minerals  

b) ETS generate “proton motive force (PMF)” that drives protons  across the membrane (to make ATP)

Highlight= important topic Underline= key fact 

3. Major classes of metabolism that use an ETS include: 

a) Organotrophy- organic electron donors  

b) Lithotrophy- inorganic electron donors

c) Photography- light excites electrons

4. Many prokaryotes use more then one type of metabolism   B. Respiratory ETS- two main examples: E. coli ETS and Mitochondrial ETS 1. Intro

a) The respiratory ETS in the bacterial cell membrane and the  mitochondrial inner membrane  

b) Microbes use many electron donors and acceptors  

(1) Aerobic: Oxygen (O2)

(2) Anaerobic: metals, oxidized ions of nitrogen, sulfur

c) A respiratory electron transport system includes at least three  functional components

(1) An initial substrate oxidoreductase (or dehydrogenase)

(2) A mobile electron carrier

(3) A terminal oxidase  

 2. E. Coli Electron Transport System 

a) The substrate dehydrogenase receives a pair of electrons from  an organic substrate, such as NADH

b) It donates the electrons ultimately to a mobile electron carrier,  such as quinone (quinone(Q) picks up 2H+ from solution and is  thus reduced to quinol: QH2)

c) The oxidation of NADH and reductio of Q is coupled to pumping  4H+ across the membrane  

d) A terminal oxidase complex, which typically includes a  cytochrome, receives the two electrons from quinol  

(1) The two H+ are translocated outside the membrane  

(2) In addition, the transfer of the two electrons through the  terminal oxidase complex is coupled to the pumping of 2H+

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e) The terminal oxidase complex transfers the electrons to a  terminal electron acceptor such as O2

(1) 2 O2 + 2H+ => 2 H2O

(2) E. Coli ETS can pump up to…

(a) 10H+ pumped across the membrane for each NADH  

molecule that is oxidized (generates an electrochemical  

gradient of protons, called a proton motor force  

(b) 6H+ for each FADH2 molecule  

 3. Mitochondrial Respiration  

a) Mitochondrial ETS differs from E. coli in these respects: (1) Possesses an intermediate cytochrome oxidase complex for  transfer of electrons

(2) Mitochondrial ETS pumps 12 H+ per NADH (two more than E.  coli)

 C. The Proton Motive Force  

1. The transfer of H+ through a proton pump gerjneates an  electrochemical gradient of protons, called a proton motive force  a) It drives the conversion of ADP to ATP through ATP synthase b) This process is known as the chemiosmotic theory

D. ATP Synthase  

1. The F1F0 ATP Synthase  

a) The F1F0 ATP synthase is a highly conserved protein complex b) The complex is made of two parts:

(1) F0- embedded in the membrane; pumps protons

(2) F1- protrudes in the cytoplasm; generates ATP

2. Harvest energy from proton motive force to synthesize ATP a) 10 protons pumped out per NADH (1 NADH produces 3  molecules of ATP)

b) 6 protons pumped out per FADH (1 FADH2 produce 2 molecules  of ATP)  

 3. Oxidative Phosphorylation

Highlight= important topic Underline= key fact 

a) ATP produced through re-oxidation of NADH and FADH2 b) Max theoretical yield: 34 ATP

(1) Glycolysis: 2 NADH => 6 ATP

(2) Transition Step: 2 NADH => 6 ATP

(3) TCA: 6 NADH => 18 ATP; 2 FADH2 => 4 ATP

4. Total ATP from One Glucose: 

a) 34 ATP from oxidative phosphorylation plus…

b) 4 ATP from substrate phosphorylation equals…

c) 38 total ATP generated (theoretical maximum)

d) -2ATP spent crossing the mitochondrial membrane for eukaryotic cells (theoretical mx of 36 ATP)

 E. Anaerobic Respiration 

1. Prokaryotes use a wide range of seminal electron acceptors,  including metals, oxidized ions of nitrogen, and sulfur

2. Anaerobic respiration generally occurs in environments where  oxygen is scarce (ex. wetland soil and human digestive tract) 3. Anaerobic reparation is unique to prokaryotes  

a) They usually possess alternative electron acceptors

b) Some bacteria use nitrate: nitrate converted to nitrite (NO3- =>  NO2-)

c) Some use sulfur compounds: (SO4^2- => SO3^3-)

F. Chapter summary  

1. Electron transport system consist of membrane-embedded proteins  that transfer electrons from an initial electron donor to a TEA that  leaves the cell

2. The ETS complexes generate a proton motive force that can drive  ATP synthesis and other cell functions  

3. Electron carriers contain metal ions and/or conjugated, double bonded ring structures  

4. An ETS includes at least three functional components:

a) Substrate dehydrogenase

Highlight= important topic Underline= key fact 

b) Mobile electron carrier 

c) Terminal oxidase

5. The F1F0 ATP synthase is a membrane-embedded protein complex;  three protons drive each F1F0 cycle, synthesizing one molecule of  ATP

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