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ENVIRONMENTAL MICROBIOLOGYENVIRONMENTAL MICROBIOLOGY
What is Microbiology?!What is Microbiology?!
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Microbiology is a branch of biology Microbiology is a branch of biology dealing with microscopic forms of life.dealing with microscopic forms of life.
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Who discovered microbiology?Who discovered microbiology?
Aristotle, Ancient Egyptians Aristotle, Ancient Egyptians and Ancient Chineseand Ancient Chinese
Robert Hooke (1635- 1703)Robert Hooke (1635- 1703)
Anton Van Leeuwenhoek (1632-1723)Anton Van Leeuwenhoek (1632-1723)
Francesco Redi (1626-1678)Francesco Redi (1626-1678)
Louis Pasteur (1822-1895) Louis Pasteur (1822-1895)
Joseph Lister (1827-1912)Joseph Lister (1827-1912)
Robert Koch (1843-1910)Robert Koch (1843-1910)
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Environmental microbiologyEnvironmental microbiology is the study of is the study of microscopic organisms found in natural microscopic organisms found in natural environments.environments.
Microbes may be eukaryotes or prokaryotes.Microbes may be eukaryotes or prokaryotes.
Bacteria, cyanobacteria, dinoflagellates, algae, and Bacteria, cyanobacteria, dinoflagellates, algae, and protozoans, are examples of microbes. protozoans, are examples of microbes.
Associated with water, soil sediments, intestinal Associated with water, soil sediments, intestinal tracts of animals, and plants.tracts of animals, and plants.
May be heterotrophic or autotrophic.May be heterotrophic or autotrophic.
Microbes live in virtually every Microbes live in virtually every environmental nicheenvironmental niche
Extremes - pH, salinity, pressure, Extremes - pH, salinity, pressure, temperaturetemperature
Aerobic, anaerobic conditionsAerobic, anaerobic conditions
BiofilmsBiofilms
Geothermal vents and subterranean Geothermal vents and subterranean depositsdeposits
Microbes make up a majority of the biomass Microbes make up a majority of the biomass on Earthon Earth
≥ ≥ 99% are not culturable at present, but may 99% are not culturable at present, but may hold valuable products for humans.hold valuable products for humans.
5-20% of biomass in seawater.5-20% of biomass in seawater.
Rich bacterial communities in sub-surface Rich bacterial communities in sub-surface strata (600 m depth) - up to 2 x 10strata (600 m depth) - up to 2 x 1044 tons- tons-equivalent to 2 m layer over entire planet. equivalent to 2 m layer over entire planet.
More than all flora and fauna combined!More than all flora and fauna combined!
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Ways to classify bacteriaWays to classify bacteriaWays to classify bacteriaWays to classify bacteria
Many Gram Negative bacteria species Many Gram Negative bacteria species are pathogenic or disease causing are pathogenic or disease causing such as:such as:
• Escherichia coliEscherichia coli• SalmonellaSalmonella• PseudomonasPseudomonas• CyanobacteriaCyanobacteria
Many Gram Positive bacteria species Many Gram Positive bacteria species are non-pathenogenic with the are non-pathenogenic with the exception of:exception of:
• Streptococcus Streptococcus • Staphylococcus Staphylococcus • Bacillus Bacillus
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ObservationsObservations• 98% of all water on Earth from marine environment.98% of all water on Earth from marine environment.
• 75% of the ocean below 1000 m and averages 3° C.75% of the ocean below 1000 m and averages 3° C.
• Deepest part of oceans ~11,000 m and ~1000 atm.Deepest part of oceans ~11,000 m and ~1000 atm.
• Many marine bacteria have absolute requirements Many marine bacteria have absolute requirements for sodium, potassium, and magnesium ions. for sodium, potassium, and magnesium ions.
• Large pressure differences lead to microorganisms Large pressure differences lead to microorganisms being present at different depths. Microbes vary inbeing present at different depths. Microbes vary in their ability to deal with pressure and salinity.their ability to deal with pressure and salinity.
• Microorganisms are involved in many geochemicalMicroorganisms are involved in many geochemical cycles in ocean waters and sediments including cycles in ocean waters and sediments including carbon, nitrogen, sulfur, and phosphorus cycles.carbon, nitrogen, sulfur, and phosphorus cycles.
ObservationsObservations• 98% of all water on Earth from marine environment.98% of all water on Earth from marine environment.
• 75% of the ocean below 1000 m and averages 3° C.75% of the ocean below 1000 m and averages 3° C.
• Deepest part of oceans ~11,000 m and ~1000 atm.Deepest part of oceans ~11,000 m and ~1000 atm.
• Many marine bacteria have absolute requirements Many marine bacteria have absolute requirements for sodium, potassium, and magnesium ions. for sodium, potassium, and magnesium ions.
• Large pressure differences lead to microorganisms Large pressure differences lead to microorganisms being present at different depths. Microbes vary inbeing present at different depths. Microbes vary in their ability to deal with pressure and salinity.their ability to deal with pressure and salinity.
• Microorganisms are involved in many geochemicalMicroorganisms are involved in many geochemical cycles in ocean waters and sediments including cycles in ocean waters and sediments including carbon, nitrogen, sulfur, and phosphorus cycles.carbon, nitrogen, sulfur, and phosphorus cycles.
Phage
Autotrophic Bacteria
Corals Corals SpongesSponges AlgaeAlgae FishFish
Dissolved Organic Matter - DOM
Protozoa
Heterotrophic Bacteria
Marine Microbial Food WebMarine Microbial Food Web
Bacteria as RemineralizersBacteria as Remineralizers
Nutrients (P,N,Fe)
Classical Marine Food WebClassical Marine Food Web
Big Algae
Herbivores
Zooplankton
Fish
Dissolved Organic Matter (DOM)Dissolved Organic Matter (DOM)
Particulate Organic Matter (POM)Particulate Organic Matter (POM)Heterotrophic BacteriaHeterotrophic Bacteria
Note:Note:
In marine environments bacteria and fungi play a In marine environments bacteria and fungi play a major role in recycling nutrients by converting major role in recycling nutrients by converting organic detritus to a more useable form. organic detritus to a more useable form.
Example: breakdown of sea grasses and Example: breakdown of sea grasses and mangrove leaves. Plant material is mostly mangrove leaves. Plant material is mostly cellulose and not easily digested by many marine cellulose and not easily digested by many marine organisms. Decomposition by microbial action organisms. Decomposition by microbial action releases phosphorus, nitrogen, and sulfur which is releases phosphorus, nitrogen, and sulfur which is in digestible form. in digestible form.
Note:Note:
In marine environments bacteria and fungi play a In marine environments bacteria and fungi play a major role in recycling nutrients by converting major role in recycling nutrients by converting organic detritus to a more useable form. organic detritus to a more useable form.
Example: breakdown of sea grasses and Example: breakdown of sea grasses and mangrove leaves. Plant material is mostly mangrove leaves. Plant material is mostly cellulose and not easily digested by many marine cellulose and not easily digested by many marine organisms. Decomposition by microbial action organisms. Decomposition by microbial action releases phosphorus, nitrogen, and sulfur which is releases phosphorus, nitrogen, and sulfur which is in digestible form. in digestible form.
Carbon cycleCarbon cycle
Phosphorus cyclePhosphorus cycle
Nitrogen cycleNitrogen cycle
Sulfur cycleSulfur cycle
• Viruses/PhageViruses/Phage 10,000,00010,000,000
• Heterotrophic BacteriaHeterotrophic Bacteria 1,000,0001,000,000
• Photosynthetic BacteriaPhotosynthetic Bacteria 100,000100,000
• ProtozoaProtozoa 4,0004,000
• AlgaeAlgae 3,0003,000
• ZooplanktonZooplankton <<1<<1
• Viruses/PhageViruses/Phage 10,000,00010,000,000
• Heterotrophic BacteriaHeterotrophic Bacteria 1,000,0001,000,000
• Photosynthetic BacteriaPhotosynthetic Bacteria 100,000100,000
• ProtozoaProtozoa 4,0004,000
• AlgaeAlgae 3,0003,000
• ZooplanktonZooplankton <<1<<1
Abundance of organisms in 1 ml of seawater
Abundance of organisms in 1 ml of seawater
• GroundwaterGroundwater 1 x 10 1 x 1044 - 4 x 10 - 4 x 1055
• Central Atlantic OceanCentral Atlantic Ocean 2 x 10 2 x 1055 - 8 x 10 - 8 x 1055
• Mediterranean SeaMediterranean Sea 2 x 10 2 x 1055 - 7 x 10 - 7 x 1055
• Antarctic coastal areasAntarctic coastal areas 2 x 10 2 x 1055- 7x 10- 7x 1055
• Coastal lagoonsCoastal lagoons 7 x 10 7 x 1055 - 2 x 10 - 2 x 1066
• Continental lakesContinental lakes 1 x 10 1 x 1066 - 43 x 10 - 43 x 1066
• Eutrophic lakesEutrophic lakes 6 x 10 6 x 1066 - 3 x 10 - 3 x 1077
• Hyperhaline pondsHyperhaline ponds up to 10 up to 1088
• GroundwaterGroundwater 1 x 10 1 x 1044 - 4 x 10 - 4 x 1055
• Central Atlantic OceanCentral Atlantic Ocean 2 x 10 2 x 1055 - 8 x 10 - 8 x 1055
• Mediterranean SeaMediterranean Sea 2 x 10 2 x 1055 - 7 x 10 - 7 x 1055
• Antarctic coastal areasAntarctic coastal areas 2 x 10 2 x 1055- 7x 10- 7x 1055
• Coastal lagoonsCoastal lagoons 7 x 10 7 x 1055 - 2 x 10 - 2 x 1066
• Continental lakesContinental lakes 1 x 10 1 x 1066 - 43 x 10 - 43 x 1066
• Eutrophic lakesEutrophic lakes 6 x 10 6 x 1066 - 3 x 10 - 3 x 1077
• Hyperhaline pondsHyperhaline ponds up to 10 up to 1088
Abundance of Microbes (cells per ml)Abundance of Microbes (cells per ml)
Direct Counts with
Epifluorescent Microscopy Direct Counts with
Epifluorescent Microscopy
Use a vacuum to pull 10 ml seawater through
a 0.02 m glass filter
Use a vacuum to pull 10 ml seawater through
a 0.02 m glass filter
Incubate with dsDNA stainIncubate with dsDNA stain
Place filter on slide Place filter on slide
Visualize on an epifluorescent microscope
Visualize on an epifluorescent microscope
Hobbie (1977) Appl Environ MicrobiolMoriarty et al (1985) Mar Biol
- 1st counts on coral reefs
Hobbie (1977) Appl Environ MicrobiolMoriarty et al (1985) Mar Biol
- 1st counts on coral reefs
Airbrush or crush coral Airbrush or crush coral
Plate onto marine agarPlate onto marine agar Extract DNAExtract DNA
Isolate 100 coloniesIsolate 100 colonies
Extract DNAExtract DNA
PCR with primers specific to 16S rDNA
PCR with primers specific to 16S rDNA
Clone and TransformClone and Transform
Pick 100 coloniesPick 100 colonies
Isolate plasmid DNAIsolate plasmid DNA
Sequence 16S rDNASequence 16S rDNA
Sequence 16S rDNASequence 16S rDNA
UnculturedUnculturedCulturedCultured
Compare 16S rDNA sequences against
each other
Compare 16S rDNA sequences against
each other
Cultured vs Uncultured Coral-Associated MicrobesCultured vs Uncultured Coral-Associated Microbes
Methods to study bacteriaMethods to study bacteriaMethods to study bacteriaMethods to study bacteria
RIBOSOMERIBOSOME
16S rDNA a gene that encodes small subunit ribosomal RNA
16S rDNA a gene that encodes small subunit ribosomal RNA
rDNA is present in all known organismsrDNA is present in all known organisms
Bact27F
V1 V2 V3
Bact517
Bact358
Bact1492R5ユ 3ユ
DNA RNA ProteinDNA RNA Proteintranscriptiontranscription translationtranslation
gradient gel electrophoresis of 16S PCR products to identify uncultured bacteria by sequencing of individual unique bands
mucus or tissue samples(multiple species)
Identifying uncultured bacteria
SummarySummary
Microbes are a key component of environment and Microbes are a key component of environment and may contain valuable compounds/info.may contain valuable compounds/info.
They are responsible for important geochemical They are responsible for important geochemical cycling.cycling.
Bacteria occupy various ecological “niches”Bacteria occupy various ecological “niches”based on their tolerance (or lack thereof) for based on their tolerance (or lack thereof) for salinities, temperatures, and baro-tolerance.salinities, temperatures, and baro-tolerance.
Many methods may be employed to study specific Many methods may be employed to study specific environmental microbiology/ecology questions.environmental microbiology/ecology questions.
SummarySummary
Microbes are a key component of environment and Microbes are a key component of environment and may contain valuable compounds/info.may contain valuable compounds/info.
They are responsible for important geochemical They are responsible for important geochemical cycling.cycling.
Bacteria occupy various ecological “niches”Bacteria occupy various ecological “niches”based on their tolerance (or lack thereof) for based on their tolerance (or lack thereof) for salinities, temperatures, and baro-tolerance.salinities, temperatures, and baro-tolerance.
Many methods may be employed to study specific Many methods may be employed to study specific environmental microbiology/ecology questions.environmental microbiology/ecology questions.
QUESTIONS?QUESTIONS?