Download Chapter nine - Growth and Metabolism v3

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
Sigler, Microbes and Society
Spring, 2013
1
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.
Sigler, Microbes and Society
Spring, 2013
2
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
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
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.
Sigler, Microbes and Society
Spring, 2013
3
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.
Sigler, Microbes and Society
Spring, 2013
4
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) doublestranded DNA.
Activity of DNAse on double-stranded DNA at different
temperatures. From: www.evrogen.com/t5.shtml
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.
Sigler, Microbes and Society
Spring, 2013
5
From: www.bact.wisc.edu/themicrobialworld/nutgro.html
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: Culture Collection of Autotrophic Organisms ( CCALA )
Institute of Botany, Academy of Sciences of the Czech Republic
Sigler, Microbes and Society
Spring, 2013
6
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.
Sigler, Microbes and Society
Spring, 2013
7
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.
Clostridium tetani as it
appears under a microscope.
Notice the “tennis racket”
shape resulting from the
shape of the spore.
10 µm
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?
Sigler, Microbes and Society
Spring, 2013
8
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.).
Sigler, Microbes and Society
Spring, 2013
9
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)
Sigler, Microbes and Society
Spring, 2013
10
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).
Helicobacter pylori is the causal agent of gastric
ulcers.
H. pylori bacteria infect the lower stomach
(antrum), causing peptic ulcer disease.
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).
Sigler, Microbes and Society
Spring, 2013
11
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?
Sigler, Microbes and Society
Spring, 2013
12
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.
Sigler, Microbes and Society
Spring, 2013
13
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.
Sigler, Microbes and Society
Spring, 2013
14
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.
Sigler, Microbes and Society
Spring, 2013
15
Energy used to drive biochemical reactions is in the form of ATP – adenosine
triphosphate.
C6H12O6
+ 6 O2
(glucose)
+ 38 ADP + 38 P à
(oxygen)
(phosphorous)
6 CO2 + 6 H2O + 38 ATP
(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.
Liberates energy
Uses energy
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.
Sigler, Microbes and Society
Spring, 2013
16
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…
Energy invested
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).
Sigler, Microbes and Society
Spring, 2013
17
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...
Energy recovered
Energy gained
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).
Sigler, Microbes and Society
Spring, 2013
18
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.
Sigler, Microbes and Society
Spring, 2013
19
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?
Sigler, Microbes and Society
Spring, 2013
20
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.
X2
Sigler, Microbes and Society
Spring, 2013
21
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.
Sigler, Microbes and Society
Spring, 2013
22
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)
Sigler, Microbes and Society
Spring, 2013
23
Some final accounting…
Each NADH generates 3 ATP
Each FADH2 generates 2 ATP
Sigler, Microbes and Society
Spring, 2013
24