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?