bio 208 - microbiology - unit 2 - lectures 7-8 - …people.cst.cmich.edu/alm1ew/208 lecture outlines...

1BIO 208 Unit 2 – Microbial Growth and Control

Unit Two – Microbial Growth and Control

In Lecture 7 we will be considering how microbes grow, what they need to be able to grow, and how they get what they need.

We will be reviewing several figures from your text. It may be helpful to have the text with you and open to Ch. 6 so you can mark those figures.

I. Microbial Growth (Chapter 6)

A. Basics“Growth” – increase in number of cells, increase in size of population.

1. Bacterial Division (Fig. 6.12, 6.14) binary fission

2. Generation Time = time required for a cell to divide (or a population to double). varies greatly with species cell number = 2n, n = # of divisions (or generations)

Ex. start with 5 cells and they each divide 9 times


B. Equation for Cell Growth

NUTRIENTS + INFORMATION + ENERGY àPOLYMERS à MACROMOLECULES à NEW CELL

source of nutrients - source of information – source of energy -

1. Energy (E):

a. E from chemicals –

1) organic (-C-C-) chemicals - chemo organo troph

Ex. C6H12O6 + 6O2 à6CO2 + 6H2O

2) inorganic chemicals – chemo litho troph

Ex. 4FeS2 + 15O2 + 14H2O à 4Fe(OH)3 + 8H2SO4

Thiobacillus ferrooxidans

b. E from light -

Ex. Chlamydomonas nivalis


2. Nutrients

a. Macronutrients: need in large quantities: Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorous, Sulfur (CHNOPS)

1). Carbonsources: CO2 – auto trophs organic (-C-C-) chemicals – hetero trophs

*note that organic chemicals can serve as both energy and carbon sources

2) Hydrogen source –

3) Nitrogensources: amino groups (-NH2) ammonia (NH3) nitrate (NO3

-) atmospheric nitrogen (N2)

4) Oxygen source –

5) Phosphoroussources: phosphate (PO4

3-) organic molecules

6) Sulfursources: sulfate (SO4

2-) sulfide (S2-)

b. Micronutrients: K, Mg, Ca, Fe required in small but sig. amts. act as cofactors for many enzymes (enz) are important in cell structures

c. Trace elements: Co, Zn, Cu required by a small number of enz.


C. Growth of Bacteria in CultureAssume cultivation in: liquid medium a closed system

affected by:o nutrient shortageso waste accumulation

Phases of Growth -4- (Fig. 6.15)

1. lag phase -

2. log phase -

3. stationary phase -


4. death phase -


D Growth of Bacteria “in the Wild”

Affected by:

1. Biofilm - A multilayered bacterial population embedded in a polysaccharide matrix and attached to some surface.

Also see Fig. 6.5

2. Cell to cell communication -

a. quorum sensing –

AHL (aka HSL) – acylhomoserine lactone

b. regulating cellular processes

Biofilm lifestyle allows these structurally simple yet physiologically diverse microbes to coexist in an environment --and the activity and growth of the community exceeds what is possible for an individual.


E. Measurement of Microbial Growth – most of this will be covered in lab, so we won’t cover much in lecture

1. Cell Numbers

a. Direct counts

advantages - quick, cheap, cell size & morph disadvantages - pop. needs to be large, can’t tell living from dead

*b. Plate counts

Diluted sample dispersed over solid agar surface - each microbe grows into 1 colony

Original # viable cells in a sample can be back calculated

Ex. dilute 1 ml sample into 100 ml water (a 1/100 dilution) à plate àincubate àcount 150 colonies

# colonies x inverse of dilution = # original per ml

150 x 100 = 15,000 CFU/ml

advantages - simple, sensitive disadvantages - have to know how to culture the microbe

Modification - counts from membrane filters

2. Cell Mass

a. turbidity – cells interfere with transmission of light, looks cloudy. The lower the light transmission, the greater the mass of cells.

b. metabolic activity – e.g., ATPase activity.

c. dry weight


In Lecture 8 we will look at the effects of the physical environment on the growth of microbes.

F. What Affects Microbial Growth?

1. Nutrientsa. presence in the environmentb. ability to transport across plasma membrane

2. Physical environment: water, pH, temp., oxygen – for each physical parameter, microbes will display a range of tolerances (too low, optimum, too high), which will vary from species to species.

optimum

too low too high


a. Water1) effects of water imbalance

hypotonic -

hypertonic -

2) adapted to hypertonic environments Halotolerant –

habitat - Ex. Staphylococcus aureus

Facultative halophile - habitat -

Extreme halophile - habitat - Ex. Halobacterium (Archaea)

b. pH internal pH of cell is

protozoa & most bact. preferfungi & algae prefer

1) effects of being in the wrong pH disrupts plasma membrane inhibits transport inactivates enz

2). pH classifications

Neutrophile: 5.5 - 8 most microbes

Acidophile: 0 - 5.5some molds, some bact.habitats - ore mines, bogs, stomachEx. Helicobacter pyloriuses for humans – enzymes to function in low pH industrial environments

Alkalophile: 8.5 - 11.5habitats - soda lakesuses – enzymes to perform in alkaline laundry detergent


c. Temperature

1) effects of being at the wrong temperature influences membrane fluidity affects enzyme functiono minimum – o optimum – o maximum -

2) temperature classifications

Psychrophile – optimum - Ex. Chlamydomonas nivalis

Psychrotroph – optimum - Ex. Listeria monocytogenes

Mesophile - optimum -Ex.

Thermophile – optimum - Ex. Thermoplasma (Archaea)

Hyperthermophile – optimum - Ex. Pyrolobus fumarii


d. Oxygen1) evolution of Earth’s atmosphere

4.8 billion no O2 (reducing = anoxic)2.25 billion oxygenic photosynthesis à O2

2 billion 1% O2

Today 21% O2

2) why is oxygen bad?

O2 - accepts e- and becomes reduced to H2O

O2 + e- à O2-

O2- + e- + 2H+ à H2O2

H2O2 + e- + H+ à H2O + OH

pull e- off of other molecules (DNA, plasma membranes)

3) what protects O2 respiring cells from bad effects of O2? -

superoxide dismutase (SOD)

2O2 + 2H+ O2 + H2O2

catalase (cat)

2H2O2 2H2O + O2

4) Oxygen tolerance classification –we will not go over in lecture but you will need to know these terms Obligate anaerobe – does not require O2 for growth, will not survive exposure

to oxygen, has neither catalase (cat) nor superoxide dismutase (SOD). Aerotolerant anaerobe – does not require O2 for growth; will survive exposure,

has SOD only. Microaerophile – needs a little O2 for growth, but less than amount present in air. Facultative anaerobe – can grow with or without O2, has both cat and SOD. Aerobe – requires O2 for growth, has both cat and SOD.

AssignmentsCh. 6 – Read entire chapterReview – 1- 3, 5-8MC – allCT – 1, 4CA – all


In Lecture 9 we will explore control of microbial growth, including the use of antibiotics.

II. Control of Microbial Growth (Chapter 7 and pp. 554-558)

A. Terminology

1. sterilization/sterilize - destroy all viable cells, spores, viruses

2. disinfection/disinfectant - kill pathogens on inanimate surfaces

3. antisepsis/antiseptic - kill pathogens on living tissue

4. de-germ – mechanical removal

5. sanitization/sanitize - lower # of pathogens to acceptable levels

B. How do we kill microbes?

1. Nonspecific – work against almost all microbes in the same way

a. Physical methods

b. Chemical methods1) phenols - denature proteins, disrupt membranesEx.

2) halogens - oxidation of cellular materialExs.

3) alcohols - denature proteins, dissolve lipid membranes -

Ex.


2. Specific – specifically kill some types of microbes, others are left unharmed.

Antibiotics (pp. 554-558)

Antibiotic –natural substance produced by one microorganism that inhibits the growth of another

a. How were antibiotics discovered?Alexander Fleming (1928)

Penicillium notatum (Eukarya – fungi)

b. How do antibiotics work?

Bactericidal - kill

Bactriostatic – inhibit

*Selective toxicity – no harm to host

**


c. Cellular Target Sites of Antibiotics – 4 - Important

1) cell wall synthesis prevent synthesis of new peptidoglycan work only on growing cells selective - how? - least toxic

Exs. penicillin, methacilllin, cephalosporin

2) plasma membrane integrity and/or function alter permeability selective - how? -

Eukarya –

Bacteria – Exs. polymyxin B, nystatin

3) nucleic acid synthesis interfere with enzymes gyrase and polymerase selective - how? -

Ex. rifampin, quinolones like ciprofloxacin

4) protein synthesis target 70S ribosome greater toxicity – why?

Exs. tetracycline, chloramphenicol, erythromycin3. Antibiotic Resistance

Fig. 20.2


a. history1940s - 1969 - 1980s - 1980s - 1990s and on -

b. how did we get in to this predicament?amount manufactured - number of prescriptions -

Antibiotic resistance can develop extremely rapidly - even in a patient receiving treatment in a hospital

Notes from clinical case:

Assignment


Read Chapter 7 and pages 554-558

Review 1, 2, 5, 7- 9MC 1,9,10CT 1-3CA 3

FYIQ. How do you know if an antibiotic is going to work?A. You should feel better within 24-48 hours of starting antibiotic treatment.

If you do not feel better:1. You have a viral infection and not a bacterial infection OR2. You have a bacterial infection but the antibiotic prescribed is not effective against the bacteria you

have OR3. You have a bacterial infection but bacteria are resistant to the antibiotic that was prescribed

Then you should contact your doctor and let her/him know that the antibiotic is not working.

What can you do to reduce the likelihood that bacteria will become antibiotic resistant?1. Take the correct dosage of your antibiotic and always take the entire prescription. If you don’t,

infectious bacteria that have not yet been killed off may survive, reproduce, and cause a more severe relapse, one that may not be treatable.

2. Ask the doctor to tailor the prescription to fit your schedule so that you don’t miss a dose.3. Ask if you can take the antibiotic for the shortest amount of time possible.4. Ask the doctor to prescribe a narrow-spectrum antibiotic, one that works specifically against a few

strains of bacteria, rather than a broad-spectrum antibiotic that targets more strains. The more bacteria exposed to antibiotics, the greater the chance that a strain will develop antibiotic resistance.

5. Use the antibiotic only for the prescribed illness. Never take antibiotics that you have left over from a previous illness. Never take antibiotics that were prescribed for someone else (not even your mom).


Review - Important Concepts for Lectures over Metabolism

I know that you have had an introduction to the basics of metabolism in BIO 110. The metabolism you learned was focused on the types of metabolism that animal and plant cells carry out --aerobic respiration. The microorganisms are tremendously more diverse and complex in metabolic patterns than are Eukarya and I want to spend our time emphasizing what microbes can do, not just covering what you have already had in other courses.

My focus in metabolism is that as microbes create and store and use energy for transporting nutrients, making their cellular components, growing, and moving in their environment. Importantly, as a consequence of their metabolism, they can profoundly change that environment!

So, if you do not remember the basics of metabolism you will need to review. The following pages should serve as a reminder. If it doesn’t all come back to you then read Chapter 5 in the text. If you have not had chemistry you will also need to read Chapter 2.

Review of nutritional patterns:Source of energy Source of carbonChemicals = chemotroph make it (CO2) = autotrophs

Organic = chemoorgano eat it (organic molecules (-C-C-C-)) = heterotrophsinorganic = chemolitho – you were not exposed to this concept in BIO 110

Light = phototroph

Most common combinations of Energy gaining strategy plus Carbon gaining strategy – this terminology was not used in BIO 110, but you were exposed to the concepts behind the terms “chemoorgano heterotroph” and “photo autotroph”)

Chemoorgano heterotrophsChemolitho autotrophsPhoto autotrophsPhoto heterotrophs

You should also know definitions of metabolism, anabolism, and catabolism

You should know what ATP is and does. ATP (Adenosine Tri Phosphate) connects reactions that produce energy with reactions that use energy. It is made to store energy for later use – it is the energy “currency” for the cell.

During catabolism – ATP à ADP + Pi + energy

During anabolism – ADP + Pi + energy à ATP

(Pi = inorganic phosphate; ADP stands for adenosine diphosphate)


ATP can be formed in 3 ways:

1. by substrate level phosphorylation – the simplest, oldest, and least-evolved way to make ATP - a high energy phosphate is removed from a substrate and is added to ADP to make ATP. Ex. C-C-C~P + ADP à C-C-C + ATP

2. by oxidative phosphorylation, aka electron transport phosphorylation – electrons are transferred from organic compounds to electron carrier molecules and then to final electron acceptor molecules. The transfer of electrons releases energy that is used to convert ADP à ATP.

3. by photophosporylation – occurs in photosynthetic cells only. Light energy is converted to ATP.

adenine

ribose

Adenosine diphosphate (ADP)

Adenosine triphosphate (ATP)

unstable bond = high energy

Adenosine monophosphate (AMP)


You should understand basics of energy productionAll molecules have energy that is associated with the electrons that form bonds between atoms. The electrons can move around in a cell from molecule to molecule, transferring energy as they move. The molecules are changed as they either gain or lose electrons.

Oxidation – Reduction Reactions (redox)

In biological systems:

H à H+ + e-

Hydrogen proton electron atom

Oxidation = a loss of an e- (and in biological systems, usually a loss of the H+ as well)Ex. H2O - 2e- - 2H+ à H2 + ½O2

Ex. NO2-- 2e- - 2H+ à NO3

-

Reduction = a gain of an e- (and in biological systems, usually a gain of the H+ as well)Ex. ½O2 + 2e- + 2H+ à H2OEx. NO3

-+ 2e- + 2H+ à NO2-+ H2O

Remember as LEO the lion says GER (Lose of Electron is Oxidation, Gain of Electron is Reduction)

Redox reactions are always balanced.

e- donor = reducing agent – causes its partner molecule to become reduced, to gain e-

e- cannot exist free in a cell, it must go somewhere. So if are e- removed from one molecule they are added to another.

e- acceptor = oxidizing agent – causes its partner molecule to become oxidized, to lose e-

In biological molecules it is usually the entire H atom (electron and proton) that is lost or gained, but not always. Sometimes the electrons are separated from the proton and only the electrons are lost or gained; and sometimes it may be one H atom + 1 electron (from a second H atom) that are lost or gained.

Ex.C3H4O3 C3H6O3 pair one

pyruvate lactic acid oxidized reduced

2e- + H+

NADH + H à NAD+ pair two reduced oxidized

NADH passes 2e- and 1 H+ to C3H4O3, as soon as C3H4O3 accepts the e-s and H+, it becomes C3H6O3 and NADH + H becomes NAD+


In any pair of molecules you can distinguish which molecule is in the oxidized state (has lost an e-) and which molecule is in the reduced state (has gained an e-):

Oxidized state Reduced state:

Contains more oxygen atoms OR Contains fewer oxygen atoms ORfewer hydrogen atoms AND more hydrogen atoms ANDtherefore has fewer electrons and is therefore has more electrons and isless negative or more positive more negative or less positive

Example pairs:

Glucose PyruvateC6H12O6 C3H4O3

NAD+ NADH

Sulfate Hydrogen sulfideSO4 H2S

End Review


In Lectures 10-13 we will explore how microbes create, store, and use energy. Through these processes microbes can profoundly change their environment.

While we will be covering the topics outlined in Chapter 5 of your text, we will be doing it in a very different manner from how it is presented in the text. Please be prepared to take careful notes in class.

III. Patterns of Metabolism in the Microbial World (a.k.a. how do microbes make a living – and why should we care?)

A. The Basics: quick review of basics from BIO 110

Metabolism = sum of all chem. rxns occurring within a living organism

All cells need a source of energy for:

Catabolism- breaking bonds in molecules –

Ex. glucose to carbon dioxide and water

Anabolism – creating bonds -


B. Patterns of Energy Production Among Living Organisms

1. Basic information

Patterns among Eukarya: alcohol fermentation (yeast) lactic acid fermentation (muscle cells, neutrophils) aerobic respiration (mold, protozoa, animals) oxygenic photosynthesis (algae, plants)

Bacteria and Archeae do all the above plus: anaerobic respiration: uses inorganic molecules other than 02 as a final electron

acceptor lithotrophy: use of inorganic substances as sources of energy photoheterotrophy: use of organic compounds as a carbon source during bacterial

photosynthesis anoxygenic photosynthesis: photophosphorylation in the absence of O2 methanogenesis: uses H2 as an energy source and produces methane light-driven nonphotosynthetic photophosphorylation: converts light energy into chemical

energy We will explore only a few of these

There are 2 initial sources of usable energy:1. sunlight – 2. chemical bonds of molecules -

Heterotrophs - energy is created by breaking bonds in a molecule and harvesting the electrons released from the H atoms in:

Organic molecules -

Inorganic molecules –

The more electrons a molecule has, the more energy it is capable of releasing. This initial molecule is called the ____________________________________________.

Ex. glucose (C6H12O6) has a lot of H atoms (12) and therefore a lot of electrons, the oxidation of glucose will release a lot of electrons. **Glucose is a high energy electron donor.


The electrons released from a molecule such as glucose have to go somewhere - they get passed from the initial donor of released electrons (electron donor) to intermediate electron carrier molecules.

Example intermediate electron carrier - NAD (Nicotinamide Adenine Dinucleotide) -

accepts 2e- (and 1 proton) and becomes reduced toNAD+ NADH

PROBLEM: NADH can’t accept anymore electrons. If energy production is going to continue, the NADH must be converted back to NAD+, which means NADH must transfer the electrons somewhere.

We will examine some of the solutions to this problem by seeing what chemoorgano heterotrophs do during carbohydrate catabolism (beginning next page)

Oxidized state

missing an H,

(which means

missing an e-)

Reduced state

(complete with e-)

NAD = Nicotinamide Adenine Dinucleotide a mobile, cytoplasm soluble electron carrier


Many types of molecules can undergo catabolism to release energy:Proteins à amino acidsLipids à glycerol + fatty acidsCarbohydrates à sugars

2. One Example – generation of energy via carbohydrate catabolism – specifically the carbohydrate glucose – by a chemoorgano heterotroph watch movement of e- and regeneration of NAD+

watch for formation of ATP (energy storage molecule)

Glycolysis (via Embden-Meyerhof Parnas (EMP) pathway) occurs in the cytoplasm – the initial electron donor is glucose

Glucose + 2 ATP 2 Pyruvate + 4 ATP net ATP production = C6H12O6 2 C3H4O3

initial e- donor


At end of glycolysis -

Need NAD+; NADH needs to get rid of e-

1. First strategy:

Fermentation –pass e- from NADH to an organic molecule, NADH becomes NAD+ - fermentation reactions occur in the cytoplasm of the cell.

organic molecule(pyruvate)2 C3H4O3 2 NADH + 2H+

2 C3H6O3 2 NAD+

(lactic acid)

End of fermentation -

Inefficient process–


2. Alternative (to fermentation) strategy:

Respiration – pass e- from NADH (becomes NAD+) along a series of intermediate electron carrier molecules, ultimately to a final (or terminal) electron acceptor molecule.

Occurs in 2 steps:

Step 1 – Tricarboxylic acid (TCA) cycle (also known as citric acid cycle or Krebs cycle) – occurs in the cytoplasm - harvests the energy still within the bonds of pyruvate, but transfers even more e- to NAD+ (so more NAD+ is converted to NADH). Doesn’t solve the shortage of NAD+ problem.

At end of TCA:

For each pyruvate (C3H4O3) à

since we get 2 pyruvate per glucose…

For each glucose à

(FAD is another intermediate electron carrier that functions so much like NAD that for the purposes of this course we will consider them equal)


Step 2 – Electron Transport Chain – the soluble NADH and FADH2 carry e- from the cytoplasm (where glycolysis took place) to the cytoplasmic membrane and pass them off to a series of membrane associated proteins (when NADH passes off the e- it becomes NAD+). These proteins function as intermediate electron acceptors, accepting e- and becoming reduced, then passing the e- off to the next protein in the chain, becoming oxidized again, ending with the final or terminal electron acceptor (which accepts the e- and becomes reduced).

This final electron acceptor may be oxygen –

Final e- acceptor (oxidized state) accepts e- and becomes reduced toaerobic respiration

1 molecule of C6H12O6 oxidized completely to CO2 coupled to reduction of oxygen to water (aerobic respiration) can yield up to a max of 38 ATP.


OR

The final electron acceptor is an inorganic molecule other than oxygen –

Examples of final e- acceptors for anaerobic respiration:

Final e - acceptor (oxidized state) becomes reduced to:

Fe3+ Fe2+ Iron respirationferric iron

NO3- NO2-, N2O, N2 Nitrate respiration

nitrate

SO42- HS- Sulfate respiration

sulfate

CO2 CH4 Methanogenesiscarbon dioxide methane

S0 HS- Sulfur respirationsulfur

Yield of ATP by cells undergoing anaerobic respiration is greater than the 2 ATP produced by glycolysis (and maintained in fermentation), but fewer than the 38 ATP produced by aerobic respiration.

anaerobic respiration


C. Some Exciting Implications of Microbial Activity:

1. Metabolism of the Human Intestinal Microbial Community

a. Where does your gut microbial community come from?

At birth

Progression of your gut community if you were a breast-fed baby

Day 1 - First colonizer was Escherichia coli facultative anaerobe chemoorgano heterotroph (gets both C and E from organic molecules)

Where did E. coli come from?

What organic compound does E. coli use as a C and E source?

How does E. coli get C and E from


galactose

Entner-Douderoff

2-keto-3-deoxygluconic acid 6-phosphate

pyruvate glyceraldehyde 3-phosphate

lactose

lactose

glucose

glucose 6-phosphate

2 pyruvate

2 acetyl CoA

lactic acidformic acid CO2 + H2

succinic acid

acetic acidethanol

CO2

CO2

Embden-Meyerhoff-Parnas

fructose 6-phosphate

2 glyceraldehyde 3-phosphate

TCA cycle

Electron transport

outside

inside

Lactose utilization by E. coli


Day 3 – 2 more bacteria joined you gut community

Enterococcusobligate but aerotolerant anaerobeschemoorgano heterotrophsobligate fermentative metabolism

Bifidobacterium

Lactic acid bacteria (named from their final fermentation end product)

e-1 Glucose NAD+

glycolysis2 ATP e-

2 Pyruvate NADH

fermentation

2 Lactic acid NAD+

Soon after added:

Enterobacter- facultative anaerobeClostridium - obligate (aerotolerant) anaerobe

Butanediol fermenters

e-1 Glucose NAD+

glycolysis2 ATP e-

2 Pyruvate NADH

fermentation

ethanol Acetoin CO2 + H2 NAD+

acetic acid lactic acid succinic acid

2,3-Butanediol + CO2


Microbial gut community from 1 week to ~ 3.5 months if you were a breast-fed infant:E. coliEnterococcusBifidobacteriumEnterobacterClostridium

3.5 months to weaning99% Bifidobacterium infantis –

When meat was introduced to your diet:

Gram-negative anaerobes:BifidobacteriumClostridiumFusobacteriumEubacteriumRuminococcusPeptococcusPeptostreptococcusBacteroides – 30% of total adult community

Bacteroides obligate anaerobeextremely oxygen sensitivefermentative metabolism


b. What are the benefits of a stable, mature gut microbial community?1) Nutritional 2) Prevents colonization by pathogens3) Trains the immune system

1) Nutritiona) Gut microbe metabolism converts complex polysaccharides to volatile fatty acids (vfa) – good

Bacteroides is the key player in this process

Host and dietary carbohydrates – complex carbs, starch, cellulose

saccharolaseshydrolases

fermentationby gut community

short-chain volatile fatty acids*

acetic acid butyric acid propionic acid

reabsorbed through the large intestine

used by you as an energy sourceprovide a significant proportion of your daily energy requirement (540 kcal)

* These products in brown are good for your health metabolic by-products

2) and 3) in supplemental info at the end of this unit, p. 38. We do not have time to cover them in this class


b) Gut microbes can metabolize dietary fats too – not so good

Bacteroides is the key player in this process also

Dietary fats

Liver

Bile acids

Absorbed by small intestine

----------------------------------------------------------------------------------------------------

If fats and bile acids are not reabsorbed by small intestine but make it to colon

deconjugated

deoxycholic acid intermediate productslithocholic acid

Bacteroides thetaiotomicron

ethyl ester

*These products in purple are mutagenic, carcinogenic products; they can induce cancer – bad for your health products


c) Gut microbes can also metabolize dietary protein – can be bad

By the combined activity of the colonic microbial community

Dietary protein

Peptides

Amino acids

Absorbed by small intestine

----------------------------------------------------------------------------------------------------

If peptides are not reabsorbed by small intestine but make it to colon

Amino acids

R

+H3N – C – C – O-

H – O

deamination decarboxylationaromatic sulfuramino acids amino acids

reduced tophenolic SO4 H2S gascompounds anaerobic respiration by

fermentation sulfate-reducing bacteria by many microbes

ammonia H2 branched chain volatile CO2

fatty acids fatty acids reduced to

CH4 gasanaerobic respiration by Methanogens (which are Archaea)

brown and purple as explained before. red are final electron acceptors in anaerobic respiration


Autotrophy - Background

All microbes need:a source of energy (electrons = ATP)a source of C to build macromolecules (-C-C-)

Heterotrophs get C to make –C-C- by recycling the C contained in organic molecules. (all heterotrophs get energy [e-, ATP] to make –C-C- from breaking chemical bonds)

**Autotrophs – get C to make –C-C- from CO2

But there are 2 sources of energy that can be used to turn CO2 à -C-C- and the source defines 2 groups of autotrophs:

1. Photo autotrophs

energy from sunlight (C from CO2) (I’ll leave this for Botany, but lots of microbes do this too)

2. Chemolitho autotrophs

energy is generated from inorganic chemicals (C from CO2)

Many different inorganic chemicals can serve as electron donors to provide the energy for microorganisms via aerobic respiration (notice the presence of O2 as electron acceptor in all the equations following – therefore all chemolitho autotrophs are obligate aerobes):

a. Hydrogen gas as an electron donor

e- donor e- acceptor donor reduced toH2 + 1/2 O2 à H2O

Hydrogen bacteriaEx. Alcaligenes faecalis (from Lab 8)

b. Sulfur compounds as electron donors

e- donor e- acceptor donor reduced to2S + 2H2O + 3O2 à 2H2SO4

Ex. Sulfur bacteria like Thiomargareta namibiensis or the bacteria that form snot-tites in caves.


c. Nitrogen compounds as electron donorsNitrifying Bacteria

2 groups of Nitrifying Bacteria:

e- donor e- acceptor donor reduced to1) 2NH3 + 3O2 à NO2 + 2H2O + 2H+

Ex. Nitrosomonas

e- donor e- acceptor donor reduced to2) 2NO2 + 2O2 à 2NO3 + 2H+

Ex. Nitrobacter

d. iron as an electron donor

e- donor e- acceptor donor reduced toFe2+ + 1/2 O2 + 2H+ à Fe3+ + H2O

Ex. Iron bacteria like Ferroplasma

Two scenarios where chemolitho autotrophs are very important:

Metabolism of Wastewater TreatmentHow do we go from toilet water to treated water? (stay tuned, we will discuss this in Unit 4 )


2. Metabolism of the Deep - What? No photosynthesis???

Deep sea hydrothermal vents – provide all the necessary inorganic chemicals Black smokers – vent hydrogen, sulfur, iron (electron donors for energy), and CO2 (for

carbon) from the Earth’s core Sea water contains dissolved oxygen (electron acceptor for aerobic respiration) Everything that is needed for chemolitho autotrophs to grow.

Chemolitho autotrophic metabolism turns CO2 and inorganic chemicals into bacterial biomass, with excess energy to spare!

Animals (chemoorgano heterotrophs)Giant tube worms

with endosymbiotic chemolitho autotrophsGiant mussels

Brittle starsLimpetsWormsCrabsVent fishSharks

AssignmentRead Chapter 5Review 4, 5b,c, 6,7,9MC 1,4,6CT 1,3,5

This ends the lecture material for Test 2.


Supplemental Information – If we have any extra time I may cover some of these topics

Aside - How can 2 people eat the same foods, 1 person gains weight and the other stays lean?

colonization of gut by microbes increases glucose uptake in the intestine

↓

microbial fermentation↓

resulting in substantial elevations in serum glucose and insulin

results in production of short-chain fatty acids

stimulate lipogenesis in the liver

↓

triglycerides into the circulation

↓

taken up by adipocytes (fat cells)

The composition and operation of your gut microbiota influences your energy balance.

Relatively high-efficiency gut microbial communities would promote energy storage (weight gain), whereas lower efficiency communities would promote weight loss.

Small but long-term differences between energy intake and expenditure can, in principle, produce major changes in body composition.

Ex. if energy intake exceeds energy expenditure by +12 kcal/day, >1 lb of fat could be gained in a year; this is the average annual weight gain experienced by Americans between ages 25 and 55.

1. b. Metabolism of Human Intestinal Microbial Community continued from p. 32

2). Mature gut microbial community prevents colonization by pathogens – pathogens like Salmonella, Shigella, Campylobacter, the pathogenic strains of E. coli, etc. that cause intestinal disease. a. competition for attachment sites – the gut epithelium is so densely colonized by normal

microbiota, nowhere for pathogens to attach.b. competition for nutrients – if pathogens do attach, they have to fight normal microbiota for a

share of nutrients

both


c. antimicrobial chemicals – and then the normal microbiota secrete antimicrobial chemicals that kill pathogens.Ex. E. coli – produces a chemical called colicin


3) Mature gut microbial community trains the immune system

The primary barrier between the outside world and you is a single layer (1 cell thick) of gut epithelium. This barrier is tight, but not impenetrable.

Microvilli – where adsorption takes place

Epithelium

Submucosa

Muscle

The surface of the intestinal epithelium is protected by your immune system – the antibody IgA, and white blood cells called T and B lymphocytes, and phagocytic macrophages.

The gut epithelium tests the contents of the gut lumen (open cavity) and can directly sense the antigens of microbes using “pattern recognition receptors”

(PRRs) – the epithelium recognizes conserved structures of bacteria and

viruses and then alerts the host to the potential of infection.

Normal microbiota of the gut and dietary antigens in food are tolerated (should not stimulate an immune response).


1. continued.c. How does what you eat influence your gut community and in turn your health? –So very cool!!

1). Over stimulation of microbial growth and metabolismEx. Lactose intolerance

In babies, the enzyme human lactase is secreted by the small intestine and will break milk lactose into glucose and galactose. By the age of weaning, humans stop secreting human lactase.

After the age of weaning if lactose is consumed in dairy, it will pass undigested to the large intestine. In the large intestine E. coli will secrete the enzyme -galactosidase, which will now break lactose in to glucose and galactose. The E. coli will use the glucose as a carbon and energy source to support rapid population growth.

As a result of their fermentative metabolism on this bounty of glucose, E. coli will produce a lot of 3 carbon fermentation end products, and a lot of CO2 gas. The 3 C end products increase the osmotic pressure in the large intestine, which combined with the CO2 will results in the symptoms of bloating and diarrhea

Adult lactose intolerance is the normal state for humans. People who as adults can tolerate lactose had ancestors that acquired a mutation that allows them to continue to secrete human lactase in to adulthood.

2). Diet can upset immune system training

The gut immune system has the challenge of responding to disease-causing microbes but not responding to food antigens and the normal gut microbial community.

In developed countries like the U.S., this discriminatory ability appears to be breaking down.

High-fat, high-sugar, low-fiber diet changes gut community composition, which upsets immune training resulting in allergies and/or chronic inflammation

Ex.1. Allergies Children w/ allergies have a higher chance of having bad Clostridium difficile and Staphylococcus aureus and lower prevalence of good Bacteroides and Bifidobacteria in their gut.

Ex. 2. Chronic inflammationCrohn's disease and ulcerative colitis (UC)

? breakdown in tolerance to Bacteroides initiates an autoimmune reaction?

Experimental txtt - whipworms


3). Diet can promote abnormal cell growth – i.e., cancer

Examples of suggested links between microbial metabolism and cancer:

1. High fat diet – go back and look at diagram of what happens to fat in the gut conjugated secondary bile acids – are carcinogens

2. High protein diet – go back and look at protein diagram again protein fermentations may be sources of systemic toxins

Heterocyclic amines (HCA) are converted into carcinogens. phenolics from aromatic amino acids may enhance production of mutagens. reduced sulfur compounds (like H2S) may be toxic to the colonic epithelium.

3. Alcohol consumption acetaldehyde toxicity

Look again at diagram of lactose utilization by E. coli. See where ethanol is produced by mixed acid fermentation? An intermediate molecule in the pathway Acetyl CoA à ethanol is a toxin called acetaldehyde

Acetyl CoA à acetaldehyde à ethanol

Part of this pathway also runs in the reverse direction:

oxidation mitochondria in the liver cellsethanol à acetaldehyde (bad) à acetic acid (good)

alcohol dehydrogenase aldehyde dehydrogenase

If there is a lot of ethanol being converted to acetaldehyde, the hepatic mitochondrial enzyme aldehyde dehydrogenase cannot keep up, and acetaldehyde levels build in the liver and blood. This causes symptoms of hangover in the short term, in the long term the acetaldehyde causes mutations in DNA that can lead to cancer.

Pr e biotics are complex carbohydrates that you cannot digest, such as fructo oligosaccharides (FOS). They pass to the intestines where they stimulate the growth and activity of intestinal bacteria that secrete beneficial metabolic end products. Fruits and vegetables contain oligosaccharides; bananas and artichokes are especially high.

Pr o biotics are living bacteria from genera that produce favorable end products, such as Bifidobacterium and Lactobacillus.


Review – Metabolism Basics

All cells need:

1. A source of carbon for making cellular molecules.

There are two strategies for obtaining carbon:a. recycle the C already present in some organic (-C-C-) moleculeb. use CO2 from the atmosphere

2. A source of energy for performing all cellular work (building molecules, transport across the plasma membrane, locomotion, etc.)

Energy is created by harvesting the electrons (e-) present in:

a. Organic molecules. (specifically the e- in the H atoms in the molecules)

Hydrogen – showing the proton and electron

like a sugar or an amino acid

OR

b. Inorganic molecules.

e- in molecules like

ammonia hydrogen sulfide

The more electrons a molecule has, the more energy the molecule is capable of yielding – so look at glucose compared to hydrogen sulfide – which molecule should yield the most energy? (glucose has 12 H vs. 2 in H2S)


The electrons that are released when bonds are broken have to go somewhere, so they get passed from the donor (the molecule that you started with that had all the electrons) to intermediate electron carriers.

NAD+ is a soluble carrier present in the cytoplasm. It is lacking 1 electron (1 H) and so it can accept 1 electron (1 H). As it accepts the electron, it is reduced to NADH.

Oxidized statefewer H, fewer e-

more positive (NAD+)

Reduced statemore H, more e-

more negative (NADH)

NAD+ is in limiting quantities in the cell and it must be converted back to NADH if energy production is to continue.

There are 2 ways convert NADH back to NAD+:

1. NADH passes the electron to an organic molecule like pyruvate – this process is called fermentation - as NADH loses the electron it becomes oxidized to NAD+ again. As pyruvate accepts the electron it becomes reduced to acetic acid or to ethanol, etc., which are excreted from the cell, carrying waste electrons with them. Acetic acid, ethanol, etc. still have electrons, so potential energy is lost in the fermentation strategy.

pH 8.5

electrochemical gradient - energyFig. 5.16


2. NADH travels to the cytoplasmic membrane and passes the electron off to the electron transport chain. This process is called respiration.

(NADH then becomes NAD+ )

The electrons are passed along the chain, generating two types of usable energy along the way – electrochemical gradient and ATP - until they reach a final electron acceptor, an inorganic molecule which can be:

a. oxygen (aerobic respiration)

OR

As oxygen accepts e- it will become reduced to H2O

b. some other inorganic molecule (anaerobic respiration)

like nitrate or sulfate

becomes reduced to nitrite (NO2)

becomes reduced to hydrogen sulfide (H2S)

Note – fermentation is NOT anaerobic respiration. By definition respiration requires both an electron transport chain and an inorganic terminal electron acceptor. Fermentation does not employ an electron transport chain and the terminal electron acceptor is an organic molecule. Fermentation takes place in the absence of oxygen, it can occur in anoxic (no oxygen but has nitrate) and anaerobic (no oxygen, no nitrate) environments, but it is not respiration!


Comparison of Respiration vs Fermentation in Chemoorganotrophs

Respiration FermentationInitial electron donor: organic molecule organic molecule

examples: carbohydrates, amino acids, lipids carbohydrates, amino acids, lipids

Intermediary electron carrier(s):

NADH, FADH2, carriers in the electron transport chain NADH

Final electron acceptor inorganic molecule organic moleculeexamples: O2 CO2, NO3, SO4 pyruvatefinal electron acceptor reduced to: H2O CH4, NO2, H2S

lactic acid, acetic acid, ethanol, etc.

example organisms Mitochondria, E. coli, Pseudomonas,

S. aureus

Methanogens, E. coli, Pseudomonas,

Sulfate-reducing bacteria

Bifidobacterium, Lactobacillus, E. coli, Clostridium,

Bacteroides

Potential net ATP yield: as many as 38 if starting with 1 glucose by aerobic respiration with an electron transport chain containing all the cytochromes – but often far fewer than 38 - but still more than 2.

2

Comparison of Respiration in Chemoorganotrophs vs Chemolithotrophs

Chemoorganotroph ChemolithotrophInitial electron donor: organic (-C-C-) molecule inorganic molecule

examples: carbohydrates, amino acids, lipids hydrogen gas, ammonia, nitrate, hydrogen sulfide

Electron donor oxidized to: CO2water, nitrate, nitrite, sulfuric

acidFinal electron acceptor inorganic molecule inorganic molecule

examples: O2 (aerobic respiration)

CO2, NO3, SO4

(anaerobic respiration)

O2 (aerobic respiration)

electron acceptor reduced to: H2O CH4, NO2, H2S H2O

example organisms

Mitochondria, E. coli, Pseudomonas,

S. aureus

Methanogens,E. coli, Pseudomonas,

Sulfate-reducing bacteria

Alcaligenes, Nitrosomonas, Nitrobacter, Thiomargarita

bio 208 - microbiology - unit 2 - lectures 7-8 - …people.cst.cmich.edu/alm1ew/208 lecture outlines...

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