chapter nine - growth and metabolism v3 · chapter 9 growth and metabolism: running the microbial...

Sigler, Microbes and Society Spring, 2013

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Chapter 9 Growth and Metabolism: Running the Microbial Machine

Objectives: After reading Chapter Nine, you should understand…

• The dynamics of a growth curve for a microbial population and identify the factors that influence the curve.

• Why microbial diversity is important to the distribution of Earth’s organisms. • The roles of enzymes and energy in microbial metabolism.

Microbes (we will focus on bacteria) have the potential for explosive growth This can be:

Problematic in the case of an infection Helpful in the case of wastewater treatment…why? Microbial growth follows predictable pattern under stable and appropriate conditions

Explained by a growth curve

§ Created by plotting the number of cells produced over time

§ Features a logarithmic cell number (y-axis)

http://www.youtube.com/watch?v=m1udFZnSukQ&feature=player_embedded


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Microbial (bacterial) growth can be described by four steps:

1. First phase – Lag Phase

No population increase is observed.

Microbes are “getting used” to the surroundings.

They synthesize cell parts and enzymes necessary to take advantage of the current conditions.

Some activity does take place, but…

Reproduction and formation of new cells is balanced by the death of other cells.

2. Second phase – Log Phase (logarithmic or exponential increase in cell number)

Microbes are at their biochemical optimum and growing and dividing at

the maximum possible rate.

Bacteria doubling (remember, bacteria divide by binary fission) occurs at regular intervals called the generation time.


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The generation time can be as short as 20 min for E. coli, but as long as days or weeks for some environmental bacteria.

Why this difference?

Because the division of bacteria is such a regular process, we can predict the number of cells accumulated during the log phase:

Xf = X0 * 2Y, where

Xf is the number of cells after Y generations;

X0 is the number of starting cells; Y is the number of generations

3. Third phase – Stationary Phase

Many microbes begin to die, which balances out the production of new ones.

Nutrients become scarce, space is at a premium, toxic waste products

accumulate.

Example problem: A pound of ground beef is contaminated with one E. coli cell and is left unrefrigerated at 37o C, the appropriate temperature for logarithmic growth. Assuming that the lag phase will last for 1 h, how many E. coli cells will be present in the ground beef after 8 h? Assume a doubling time of 20 min and no stationary or death phase.

The solution:

Number of hours of logarithmic growth = 8 h – 1 h = 7 h.

Minutes of logarithmic growth = 7 h * 60 min/h = 420 min. 420 min/20 min = 21 doublings (generations) = Y

Xf = X0 * 2Y

Xf = 1 * 221

Total number of cells after 8 h (21 generations) = 2.1 x 106 cells


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4. Final phase – Death Phase

Population size rapidly decreases because cells are dying faster than they can be produced.

Some bacteria might begin to produce spores in order to survive

Each species of microbe has a unique growth curve, but all are characterized by the same

phases. What if growth was uncontrolled with unlimited resources for 36 hours?

Assume a doubling time of 20 minutes and an average cell volume of 1 x 10-18 m3 (1 µm x 1.5 µm). Starting with one E. coli, the number of cells present after 36 h will be 2108 (108 doublings in 36 h) = 3.2 * 1032 E. coli. This results in a total cellular volume of 3.2 * 1014 m3. Assuming the Earth’s surface area (including water) is 5 * 1014 m2, the E. coli would spread over the Earth in a layer about 0.6 m thick.

Of course this doesn’t happen, even though we often grow bacteria in the laboratory for much longer than 36 hours. Why?

Many environmental parameters impact the growth and metabolism of microbes

1. Water

Water is necessary for microbial metabolism to occur.

Only spores can survive extended dry conditions

Quite simply, water allows biochemical reactions to occur.


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2. Temperature

Temperature impacts the rates of biochemical reactions by influencing the rates of enzyme activity.

If temperatures are too low, reaction rates are reduced.

If temperatures are too high, breakdown (denaturation) of the enzymes will limit activity.

Example: DNase – an enzyme that breaks down (digests) double-

stranded DNA. Temperature will also impact the composition of the microbial community.

The high diversity of microorganisms has allowed for them to occupy

almost all of Earth’s environments. Microorganisms are adapted to a wide range of temperatures.

Activity of DNAse on double-stranded DNA at different temperatures. From: www.evrogen.com/t5.shtml


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Where might each of these groups be found?

A. Psychrophiles (prefer temperatures up to 20o C)

e.g., Chlamydomonas nivalis, a single celled green algae. C. nivalis’ red color comes from to a bright red pigment that protects the algae from intense radiation. The pigment also absorbs heat, providing the algae with liquid water as the snow melts around it.

From: www.bact.wisc.edu/themicrobialworld/nutgro.html

From: Culture Collection of Autotrophic Organisms ( CCALA ) Institute of Botany, Academy of Sciences of the Czech Republic


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Psychrophiles also grow well in the refrigerator.

The spoiling of milk is initiated by Streptococcus spp. Streptococci convert the milk sugar (lactose) to lactic acid.

Not a danger to your health, but undesirable.

Staphylococcus spp. can grow in refrigerated foods and produce

toxins.

Effects of eating this food can be severe.

How do you prevent these effects? Hint: toxins are proteins.

B. Mesophiles (prefer temperatures between 20 – 45o C)

Microbiologists once believed that most of the microorganisms on the planet were mesophiles, but more and more organisms that live at extreme temperatures are being discovered.

By looking at the temperatures at which mesophiles grow, you should be

able to tell me what organism might serve as a very important reservoir/host for mesophilic pathogens?

C. Thermophiles (45o C and above)

We discussed examples of these (Archaea) in Chapter Five. Overall, temperature selects for those organisms whose enzymes can

handle heat or cold.

Thermus aquaticus is a bacterium that can tolerate high temperatures. Because of their durability, enzymes from T. aquaticus are used in laboratory protocols that are performed under high temperatures.


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3. Oxygen Four classes of organisms exist with respect to their use of oxygen:

A. Most known species of microbes are aerobic – they require oxygen for

their metabolism. (More later)

B. Anaerobic microbes require an absence of oxygen and will die if any oxygen is present.

Some pathogens are anaerobic.

Clostridium tetani – spores are present in soil and in animal feces.

When spores enter the skin through a puncture site, they can germinate and the resulting bacteria can generate powerful toxins in the anaerobic regions of the wound.

Toxins cause prolonged contraction of skeletal muscle fibers (lockjaw).

Tetanus is often associates with puncture wounds from rusty metal…why?

Clostridium tetani as it appears under a microscope. Notice the “tennis racket” shape resulting from the shape of the spore.

10 µm


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Anaerobes can be difficult to work with in the lab…

C. Facultative microbes can grow in the presence or absence of oxygen.

Example: intestinal bacteria

Many fecal pathogens are facultative, which means they can live in the anaerobic environment of the primary host (source human or animal), then in the aerobic secondary environment (beach, stream, sand), and then finally in a newly-infected host (another human).

D. Microaerophilic microbes grow best in oxygen-poor environments, but still require oxygen. (usually 2 – 10%)

Deep soils Intestinal and urinary track

Most urinary tract infections are caused by a small range of bacteria (E. coli, Proteus spp., Enterococci, Shigella spp.).


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4. Acidity or alkalinity (pH)

What is pH?

Most microbes have an optimum pH at which they prefer to grow and metabolize.

Partially dictated by the pH optimum of the microbe’s enzymes.

Example: DNase

DNase activity at differing pH. From Hsiang

et al. Biochem. J. (1998) 330 (55–59)


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Most bacteria grow well at neutral pH (7.0), but can span a range from 5.0 – 8.0.

Some extreme microbes can grow at much lower pHs (remember the bacterium Sulfolobus acidocaldarius?).

These are called acidophiles

Example: Helicobacter pylori causes 70% of gastric ulcers and 90% of duodenal ulcers (pH between 1 – 3).

H. pylori do not actually invade the cells of the stomach but rather

release chemicals that the stomach tissues absorb. These chemicals cause inflammation of the tissues that can

develop chronic active gastritis, peptic ulcer disease, or gastric cancer (because the gastric cells are affected).

Helicobacter pylori is the causal agent of gastric ulcers.

H. pylori bacteria infect the lower stomach (antrum), causing peptic ulcer disease.


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Microbial Metabolism

From the Greek word metaballein = “change”.

Thousands of reactions are occurring in a microbe at any one time, but they fall into two broad groups:

1. Biosynthesis reactions (anabolic) – a chemical process that results in the formation of cellular structures and molecules.

Usually consumes energy.

2. Digestive reactions (catabolic) – large molecules or compounds are broken down into smaller ones.

Usually results in the liberation of energy.

The reactions that govern the growth and metabolism of microbes are only possible because of special proteins called enzymes.

Like all proteins, enzymes are long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties. Enzymes are catalysts for metabolic reactions – they speed up the reaction, but don’t change themselves.

They are not consumed in the reaction, so they are available to speed up

multiple reactions. Without the enzyme, the reaction would still take place, but would require a much

longer time (instead of seconds, maybe days, months or even years). In what situation might this be a problem?


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Enzymes catalyze very specific substrates that are involved in these reactions.

The function of lysozyme (right) is to hydrolyze (break) the bonds between residues of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). These are the sugars in peptidoglycan.

Example: breakdown of carbohydrates.

Think of a rusty nut and bolt…adding oil (like an “enzyme”) will lower the amount of energy needed to loosen it.


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The mechanics of enzyme activity:

Over 2000 enzymes have been named. Each name has two parts:

1. A prefix that reflects the substrate that the enzyme acts on.

2. The suffix –ase.

Examples:

Lactase, for example, breaks down lactose. Lipase breaks down lipids. Helicase unwinds the double helix of DNA.

DNase breaks down DNA.


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Producing Energy The major goal of metabolism is to provide energy to the organism. What is this energy used for? Energy cannot be created, only converted from one form to another. Carbon is a central element the makes up many compounds used as energy sources by microbes.

The carbon cycle highlights many of the conversion reactions that take place to provide energy.

Plants and some microbes trap sunlight to generate energy that combines the carbons from CO2 to synthesize carbohydrates (like glucose) – Photosynthesis

“Sunlight energy” (light) is converted to “carbohydrate energy” (apple).

These carbohydrates (in the form of sugars in the plant and bacteria biomass) and other energy-rich compounds (proteins, lipids) are used by heterotrophic organisms (some bacteria, cows, humans, etc.) to generate energy to drive metabolic reactions.


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Energy used to drive biochemical reactions is in the form of ATP – adenosine triphosphate.

C6H12O6 + 6 O2 + 38 ADP + 38 P à 6 CO2 + 6 H2O + 38 ATP (glucose) (oxygen) (phosphorous) (carbon dioxide) (water) (energy)

The process of converting these energy sources to ATP is called respiration.

ATP is like a portable battery that provides a universal currency of energy.

Why do microorganisms need this energy?

ATP is used as a currency of energy for all organisms.

ATP is actually a nucleotide (like the ones used to build DNA). Much of the energy in ATP is released when the bonds connecting the

third phosphate is broken by enzymes. This creates ADP, adenosine diphosphate.

Uses energy

Liberates energy


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Respiration and glycolysis Let’s use glucose as an example of how microbes can gain energy in the form of ATP.

Keep in mind that many other molecules other than glucose can be used to generate energy using these same reactions (important for the alcoholic beverage industry, as we will see).

The breakdown of glucose occurs by the process of glycolysis (glyco = sugar; lysis = to break). During glycolysis, glucose (6 carbons) is converted to a smaller, 3-carbon compound

called pyruvate. Glycolysis occurs in the cytoplasm of microbes and does not require oxygen.

The goal of glycolysis is to make usable energy in the form of ATP from a molecule of glucose.

Let's break it down… Preparatory steps: the first “half” of the glycolysis pathway…

A few things are of key importance in the first “half” of the glycolytic pathway:

1. ATP is used-up in two preparatory reactions to “energize” the glucose (step 1 and step 3).

Energy invested


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These ATPs will be recovered in later reactions.

2. In step 4 an important split takes place.

A six-carbon molecule is split into two, three-carbon molecules. This is important, because now everything that happens in subsequent

steps happens twice.

Recovering and gaining energy: the second “half” of glycolysis...

3. At step 5, electrons are stripped from the 3-carbon molecule and attached to NAD, an electron carrier, along with a proton (H+).

This makes NADH, which will become very important later (remember,

since the split took place during step 4, this results in two NADHs).

Energy gained

Energy recovered


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These will become important later.

4. In step 6, a phosphate is released from the 3-carbon molecule and added to

ADP to form ATP. This occurs twice, because of the split at step four, resulting in 2 ATPs. Represents the recovery of the energy invested during steps 1 and 3.

5. In step 9, another phosphate is released from the 3-carbon molecule and added to another ADP to form two more ATPs.

This results in the generation of two pyruvates. Represents the gain of energy (this is why glycolysis is performed by all

organisms).

So, overall, two ATPs were invested in the beginning of glycolysis, two were recovered in the middle, and another two gained at the end.

Results in the net gain of two ATPs to use for energy in the cell. Fermentation Thus far, the glycolysis pathway we have discussed has not required oxygen to gain

energy from glucose. If oxygen is available, pyruvate will be used to gain even further energy from the

Krebs cycle (later). In the continued absence of oxygen, fermentation can take place. Pyruvate is converted to alcohols, acids and CO2.

This is what yeast does when used for brewing, producing ethyl alcohol.


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Notice that NAD is regenerated by the yeast to be used in further glycolysis.

Fermentation is of vital importance to the alcohol industry. The type of beverage produced is dependent on the starting material for

glycolysis: Grapes - leads to wine Barley – leads to beer Potatoes – leads to potato wine and ultimately, vodka

The carbon dioxide (CO2) that is produced makes the carbonation in beer and champagne.

Acids can also be produced by fermentation, namely by bacteria.

Lactobacillus spp. and Streptococcus spp. Where have we seen these two bacteria before?


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The Krebs cycle While fermentation occurs in the absence of oxygen (O2), the presence of oxygen can

allow aerobic microbes to gain much more energy (in the form of ATP). The Krebs cycle is an intimidating, complex pathway that was characterized in the 1950s.

It is a cycle, which means that the end product can be used to start the pathway again.

The cycle picks up where glycolysis leaves off; with pyruvate.

Before the cycle starts though, pyruvate gets converted to acetyl CoA, releasing CO2 in the process (this is another CO2 produced when organisms breathe = respiration).

Again, electrons are stripped from the pyruvate and attached to NAD along with a proton (H+).

This makes another NADH, which will become very important later.

X 2


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Now the Krebs cycle can begin:

We will not go into detail but several features need to be pointed out.

1. No ATP is used in the Krebs cycle.

How is this different from glycolysis?

2. The cycle turns two times for every glucose that was broken down during glycolysis (remember, because two, 3-carbon pyruvates enter the cycle).

3. Two ATPs are produced directly from the cycle (1 ATP for each turn).

4. NADH and FADH2 are produced during four steps (C, D, E and G). I promise to reveal the importance of this shortly.

5. Four CO2 are produced.

So, the Krebs cycle further processes the broken-down glucose (as pyruvate) to produce another two ATP. How much work can a bacterium do with so few ATP? Think about this: A person needs (burns) 50 calories to walk for 15 min. Each molecule of glucose provides about 10-21 calories of energy…so, 50/10-21 =

5x1022 molecules of glucose needed to walk for 15 min. Each molecule of glucose provides 38 ATP, so 38 ATP x 5x1022 = 2x1024 ATP

needed to walk for 15 min.

Glycolysis and the Krebs cycle produce four ATPs. Doesn’t this seem like a lot of steps just to generate four ATPs?? It does to me.

Where do the other 34 ATP per glucose come from?

Certainly we can gain more than FOUR ATPs from these cycles: The electron transport system and chemiosmosis. The processes of glycolysis and the Krebs cycle represent the first two phases of energy

generation (respiration) by microbes. The electron transport system (ETS) is the third.


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The ETS occurs in the cell membrane of bacteria. (It occurs in the mitochondrion of eukaryotes).

The NADH and FADH2 that we have discussed previously are used in the ETS to

generate even more ATP.

Special molecules called electron carriers act as a “bucket-brigade” passing electrons (potential energy) between each other.

The steps: Electrons are sequentially passed from NADH and FADH2 to the electron carriers

(cytochromes).

Protons (H+) are split from the NADH and FADH2 and are moved from inside the membrane to the outside of the membrane.

Results in a high relative concentration of H+ outside the

membrane. When enough H+ have accumulated to create an unbalanced amount of H+

inside and outside of the cell, an equilibrium occurs and the H+ rush back through the membrane.

On the way back into the cell, H+ are directed through an ATP

synthetase, which adds P to ADP, resulting in the production of ATP.

(Chemiosmosis)


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Some final accounting… Each NADH generates 3 ATP Each FADH2 generates 2 ATP

chapter nine - growth and metabolism v3 · chapter 9 growth and metabolism: running the microbial...

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