Download Environmental microbiology File

CEE 160L: Introduction to
Environmental Engineering and
Science
Lectures 3 and 4 – Environmental Microbiology
Microbiology tutorial
• http://classroom.sdmesa.edu/eschmid/Lectur
e4-Microbio.htm
Outline today’s lecture
 Different Kinds of Microbes
 Cell Structure
 Observing Microbial Growth
 Different types of microscopy
 Quantifying Microbial Growth
Different Classes of Microbes
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Bacteria
Archae
Fungi
Protists
Viruses (NOT MICROBES)
Mammalian
cells
=Bacteria + Archaea
Bacteria
Bacteria fun facts
• Inhabit soil, water, acidic hot springs, radioactive
waste, and the deep portions of Earth's crust.
• Can live off sunlight and most inorganic and organic
substrates
• Biomass exceeds that of all plants and animals
• Vital in recycling nutrients (e.g nitrogen from the
atmosphere)
• Important in sewage treatment and the breakdown of
oil spills, the production of cheese and yogurt through
fermentation, antibiotics manufacture
Not fun bacteria facts
• People have ~10x more bacterial cells than human cells
• Widespread use of antiobiotics has caused bacteria to
develop resistant strains
• Tuberculosis alone killing about 2 million people a year,
mostly in sub-Saharan Africa
Deinococcus radiodurans is the most
extreme of extremophiles
Nicknamed Conan the Bacterium
D radiodurans fun facts
• First half of name is Greek for “terrible berry” the
second half is Latin for “radiation surviving”
• Able to withstand DNA damaging radiation does of
5000Gy (exposing 100 people to this does would kill
~50)
• Resistance from having multiple copies of its genome
and rapid DNA repair mechanisms
• Been genetically engineered for use in bioremediation
to consume and digest solvents and heavy metals, even
in a highly radioactive site
20000mSV=
20Sv=20Gy
Sterilization dose:
25kGy
Archae
“WEIRD BACTERIA”
Archaea fun facts
• Visually similar to bacteria, but possess genes and
several metabolic pathways and structures that are
more common to eukaryotes
• Widespread like bacteria (including in/on humans).
Many in extreme environments.
• Important to Earth’s life cycle and human digestion
• Do not seem to be pathogenic
• Used in biogas production and sewage treatment
• Some have very strange shapes
Fungi
Fungi fun/un-fun facts
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Eukaryotes
Yeasts. molds, mushrooms
Important in decomposition of organic matter
Production of antibiotics, and, more recently, various
enzymes produced by fungi are used industrially and in
detergents
• Cause crop losses and food spoilage (matooke blight)
• Inconspicuous as filaments except when fruiting
(mushrooms, mold)
• Can be very toxic (mushroom misidentification)
Protists
Protist fun facts
• Unicellular or multicellular organism without
specialized tissues found in plants
• Often classified by appearance: "animal-like" protozoa,
the "plant-like" protophyta (mostly unicellular algae),
and the "fungus-like" slime molds and water molds
• Many are photosynthetic and are vital primary
producers in ecosystems: algae and plankton
• Pathogenic species such as the kinetoplastid
Trypanosoma brucei, which causes sleeping sickness
and species of the apicomplexan Plasmodium which
cause malaria
Viruses
Virus un-fun facts
• Virus is Latin for poison
• Not considered alive
• Infectious agent that replicates only inside the living
cells of other organisms.
• Can infect all types of life forms, including bacteria and
archaea
• Cause of HIV/AIDS and Ebola
Two Cell Structures
 Prokaryotes:
Simple Structure
Eukaryotes:
Complex Structure (membrane-enclosed
organelles).
Prokaryotic Cell Components
 Cell Membrane
 Cell Wall
 Cytoplasm
 Capsule
 Nuclear Region
 Ribosomes
 Plasmids
 Flagella
Prokaryotic Cell Structure
• Bacteria
• Archae
Eukaryotic Cell Layout
• Fungi
• Protists
Virus vs. Bacteria
What are cells made of?
Element
C
O
Source
Function
Organic compounds, CO2
(sugars, fats, proteins)
O2, H2O, organic cmpds, CO2
Main cell constituents
H
H2, H2O, organic cmpds
N
NH4, NO2, N2, proteins
S
SO2-, HS-, S, S2O32-, proteins
organic sulfur compounds
In Cys, Met, T, biotin
CoA
P
HPO42-
In nucleic acids &
phospholipids
This & next 2 tables based on G. Gottschalk, Bacterial Metabolism, 2nd Ed., p.2-3, 1986
Major Inorganic Elements
Element
K
Source
K+
Function
Primary inorganic cation,
Required in a variety of
enzymes.
Mg
Mg2+
enzymes
Required in a variety of
Stabilizes cell walls, membranes,
Ca2+
in exoenzymes and cell walls,
important in spores (heat
ribosomes
Ca
stability)
Fe
Fe2+, Fe3+
In cytochromes, ferredoxins,
Iron-sulfur proteins, cofactor
of some enzymes
Na
Na+
Required by some but not all organisms
Cl
Cl-
Important cellular anion
Minor Elements
Element Source Function
Zn
Zn2+
Alcohol dehydrogenase, alkaline
phosphotase, aldolase, RNA and DNA
polymerase
Mn
Mn2+
Superoxide dismutase, photosystem II,
cofactor of PEP carboxykinase
Mo
MoO42- Nitrate reductase, nitrogenase, xanthine
dehydrogenase, formate dehydrogenase
Se
SeO32- Glycine reductase, formate
dehydrogenase
Co
Co2+
Coenzyme B12 enzymes
Cu
Cu2+
Cytochrome oxidase, nitrite reductase,
Ni
Ni2+
Urease, hydrogenase, factor F430
W
WO42-
Some formate dehydrogenases
What are cells made of?
Component % wt
mol. wt
# per cell # of kinds
H2O
70
18
4 x 1010
1
Inorg. ions
1
40
2.5 x 108
20
Carbohydrates 3
150
2 x 108
200
Amino acids
0.4
120
3 x 107
100
Nucleotides
0.4
300
1.2 x 107
200
Lipids
2
750
2.5 x 107
50
Other small mol.0.2
150
1.5 x 107
200
Proteins
15
40,000
106
2-3000
DNA
1 2,500,000,000
4
1
RNA:
6
rRNA
0.5-1,000,000
6 x 104
2
tRNA
25,000
4 x 105
40
mRNA
1,000,000
103
1000
J.D. Watson, Molecular Biology of the Gene, Table 3.3, 1970
Macromolecules: 95% of dry weight
Macromolecule
wt %
protein
52.4 (50 to 80%)
polysaccharide
16.6 (12 to 28%)
lipid
9.4 (8%)
RNA
15.7
DNA
3.2
15 to 25%
A.H. Stouthamer, Antonie van Leeuwenhoek 39, 545-565 (1973)
Empirical Formulas
• For Organic Portion
• C5H7O2N = CH1.4O0.4N0.2
• CH1.9O0.3N0.24P0.02Na0.01S0.007K0.006Mg0.005
Cl0.003Fe0.001 reported for E. coli
Metabolic Characterization of
Microorganisms
• Carbon Source
– Organic carbon - heterotrophs
– Inorganic carbon (CO2) - autotrophs
• Energy Source
– Organic chemicals – chemoorganotrophs
• Carbon based compounds other than CO2: Sugars
(glucose), Gasoline, etc.
– Inorganic chemicals – chemolithotroph
• NH3, Fe2+, CO2, H2, S0
– Light (photosynthesis) - phototrophs
How do cells get their energy?
Electrons are transferred through a
series of redox reactions that creates
a “proton motive force” utilized to
generate ATP (adenosine triphosphate). ATP is the primary
energy form in cells.
Metabolic Characterization
• Electron Acceptor (electrons are shuttled
via electron carriers to the terminal
electron acceptor)
– Aerobic – Require Oxygen
– Anaerobic – Use something other than oxygen
or carry out fermentation
Examples: NO3-, SO4-2, CO2
CH3COOH
CO2 + CH4
Metabolic Characterization
• Electron Acceptor
– Obligate Anaerobes – require complete
absence of O2 because it is toxic to them
Example: methanogens
– Facultative – can grow without oxygen or in
low oxygen environments
• Will use oxygen if present; otherwise another
electron acceptor is used.
Metabolic Characterization
Examples
Aerobic conventional heterotroph
Carbon & Energy Source: Organic Carbon
Electron Acceptor: Oxygen
Facultative conventional heterotroph
Carbon & Energy Source: Organic Carbon
Electron Acceptor: Oxygen or another electron acceptor
Metabolic Characterization
Examples
Aerobic chemolithotroph
Energy Source: Inorganics
Carbon Source: Inorganics
Electron Acceptor: Oxygen
How to observe microbes?
• Light Microscopy
– Bright Field, Dark Field and Phase Contrast
• Electron Microscopy
– Transmission Electron Microscopy
– Scanning Electron Microscopy
Light Microscopy
3-D Imaging – Interference contrast
and Atomic Force Microscopy
Confocal Microscopy
Electron Microscopy
Shapes of Bacteria
• Coccus
– Chain = Streptoccus
– Cluster = Staphylococcus
• Bacillus
– Chain = Streptobacillus
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Coccobacillus
Vibrio = curved
Spirillum
Spirochete
Chapter 4
Cell Growth

Binary Fission
(Prokaryotes)
• Budding (Fungi)
http://mpf.biol.vt.edu/research/budding_yeast_model/p
p/intro.php
Measuring Microbial Growth
Direct Observation or Direct Counting
Observe or Count cells in known volume of
sample microscopically
Cells often hard to see, so some sort of
staining procedure commonly used
Two classes of stain:
Specifically stain nucleic acids or proteins by adsorption
onto these materials
Stains that react (fluoresce) as a result of metabolic
activity in the cell
Viable Counts
• Spread a known volume of diluted sample on an
agar plate and count colonies after incubation.
– Assumes each colony resulted from a single cell
• Reported as colony forming units (CFU)/volume
(e.g., mL)
• Major limitation: Selectivity!
– Although sometimes an advantage (e.g., specific
organism tests)
• For Environmental samples, microscopic stain
counts are usually 102 to 104 times the viable
count.
Biochemical Methods
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Measure
Measure
Measure
Measure
Measure
Measure
Total Cell Protein
Nucleic Acids
ATP
Chlorophyll (use for algae)
enzyme activity
respiration:
– Oxygen uptake
Microbial Biomass
• Dry weight of cells (e.g., VSS)
• Turbidity
– Rapid measurement that can be correlated in
some cases to other techniques (e.g., dry
weight)
Questions
• Who is the audience?
• What is the premise of the study? Why do it?
• What was their approach?
• What did they observe?