1. ENVIRONMENTAL MICROBIOLOGY

2. What is Microbiology?!

3. Microbiology is a branch of biology dealing with microscopic forms of life.

4. Who discovered microbiology? Aristotle, Ancient Egyptians and Ancient Chinese Robert Hooke (1635- 1703) Anton Van Leeuwenhoek (1632-1723) Francesco Redi (1626-1678) Louis Pasteur (1822-1895) Joseph Lister (1827-1912) Robert Koch (1843-1910)

5. Environmental microbiology is the study of microscopic organisms found in natural environments. Microbes may be eukaryotes or prokaryotes. Bacteria, cyanobacteria, dinoflagellates, algae, and protozoans, are examples of microbes. Associated with water, soil sediments, intestinal tracts of animals, and plants. May be heterotrophic or autotrophic.

6. Microbes live in virtually every environmental niche Extremes - pH, salinity, pressure, temperature Aerobic, anaerobic conditions Biofilms Geothermal vents and subterranean deposits

7. Microbes make up a majority of the biomass on Earth ≥ 99% are not culturable at present, but may hold valuable products for humans. 5-20% of biomass in seawater. Rich bacterial communities in sub-surface strata (600 m depth) - up to 2 x 10 4 tons- equivalent to 2 m layer over entire planet. More than all flora and fauna combined!

9. Ways to classify bacteria

10. Many Gram Negative bacteria species are pathogenic or disease causing such as: Escherichia coli Escherichia coli Salmonella Salmonella Pseudomonas Pseudomonas Cyanobacteria Cyanobacteria Many Gram Positive bacteria species are non-pathenogenic with the exception of: Streptococcus Streptococcus Staphylococcus Staphylococcus Bacillus Bacillus

12. Observations 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 in being 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 geochemical Microorganisms 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.Observations 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 in being 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 geochemical Microorganisms 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.

13. Phage Autotrophic Bacteria Corals Sponges Algae Fish Dissolved Organic Matter - DOM Protozoa Heterotrophic Bacteria Marine Microbial Food Web

14. Bacteria as Remineralizers Nutrients (P,N,Fe) Classical Marine Food Web Big Algae Herbivores Zooplankton Fish Dissolved Organic Matter (DOM) Particulate Organic Matter (POM) Heterotrophic Bacteria

15. Note: In marine environments bacteria and fungi play a major role in recycling nutrients by converting organic detritus to a more useable form. Example: breakdown of sea grasses and mangrove leaves. Plant material is mostly cellulose and not easily digested by many marine organisms. Decomposition by microbial action releases phosphorus, nitrogen, and sulfur which is in digestible form. Note: In marine environments bacteria and fungi play a major role in recycling nutrients by converting organic detritus to a more useable form. Example: breakdown of sea grasses and mangrove leaves. Plant material is mostly cellulose and not easily digested by many marine organisms. Decomposition by microbial action releases phosphorus, nitrogen, and sulfur which is in digestible form.

16. Carbon cycle

17. Phosphorus cycle

18. Nitrogen cycle

19. Sulfur cycle

20. Viruses/Phage10,000,000 Viruses/Phage10,000,000 Heterotrophic Bacteria1,000,000 Heterotrophic Bacteria1,000,000 Photosynthetic Bacteria100,000 Photosynthetic Bacteria100,000 Protozoa 4,000 Protozoa 4,000 Algae 3,000 Algae 3,000 Zooplankton<<1 Zooplankton<<1 Viruses/Phage10,000,000 Viruses/Phage10,000,000 Heterotrophic Bacteria1,000,000 Heterotrophic Bacteria1,000,000 Photosynthetic Bacteria100,000 Photosynthetic Bacteria100,000 Protozoa 4,000 Protozoa 4,000 Algae 3,000 Algae 3,000 Zooplankton<<1 Zooplankton<<1 Abundance of organisms in 1 ml of seawater

21. Groundwater 1 x 10 4 - 4 x 10 5 Groundwater 1 x 10 4 - 4 x 10 5 Central Atlantic Ocean 2 x 10 5 - 8 x 10 5 Central Atlantic Ocean 2 x 10 5 - 8 x 10 5 Mediterranean Sea 2 x 10 5 - 7 x 10 5 Mediterranean Sea 2 x 10 5 - 7 x 10 5 Antarctic coastal areas 2 x 10 5 - 7x 10 5 Antarctic coastal areas 2 x 10 5 - 7x 10 5 Coastal lagoons 7 x 10 5 - 2 x 10 6 Coastal lagoons 7 x 10 5 - 2 x 10 6 Continental lakes 1 x 10 6 - 43 x 10 6 Continental lakes 1 x 10 6 - 43 x 10 6 Eutrophic lakes 6 x 10 6 - 3 x 10 7 Eutrophic lakes 6 x 10 6 - 3 x 10 7 Hyperhaline ponds up to 10 8 Hyperhaline ponds up to 10 8 Groundwater 1 x 10 4 - 4 x 10 5 Groundwater 1 x 10 4 - 4 x 10 5 Central Atlantic Ocean 2 x 10 5 - 8 x 10 5 Central Atlantic Ocean 2 x 10 5 - 8 x 10 5 Mediterranean Sea 2 x 10 5 - 7 x 10 5 Mediterranean Sea 2 x 10 5 - 7 x 10 5 Antarctic coastal areas 2 x 10 5 - 7x 10 5 Antarctic coastal areas 2 x 10 5 - 7x 10 5 Coastal lagoons 7 x 10 5 - 2 x 10 6 Coastal lagoons 7 x 10 5 - 2 x 10 6 Continental lakes 1 x 10 6 - 43 x 10 6 Continental lakes 1 x 10 6 - 43 x 10 6 Eutrophic lakes 6 x 10 6 - 3 x 10 7 Eutrophic lakes 6 x 10 6 - 3 x 10 7 Hyperhaline ponds up to 10 8 Hyperhaline ponds up to 10 8 Abundance of Microbes (cells per ml)

22. 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 stain Place filter on slide Visualize on an epifluorescent microscope Visualize on an epifluorescent microscope Hobbie (1977) Appl Environ Microbiol Moriarty et al (1985) Mar Biol - 1 st counts on coral reefs Hobbie (1977) Appl Environ Microbiol Moriarty et al (1985) Mar Biol - 1 st counts on coral reefs

23. Airbrush or crush coral Plate onto marine agar Extract DNA Isolate 100 colonies Extract DNA PCR with primers specific to 16S rDNA Clone and Transform Pick 100 colonies Isolate plasmid DNA Sequence 16S rDNA Uncultured Cultured Compare 16S rDNA sequences against each other Compare 16S rDNA sequences against each other Cultured vs Uncultured Coral-Associated Microbes

24. Methods to study bacteria

25. RIBOSOME 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 organisms Bact27F V1V2 V3 Bact517 Bact358 Bact1492R 5 ﾕ 3 ﾕ DNA RNA Protein transcription translation

26. 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

27. Summary Microbes are a key component of environment and may contain valuable compounds/info. They are responsible for important geochemical cycling. Bacteria occupy various ecological “niches” based on their tolerance (or lack thereof) for salinities, temperatures, and baro-tolerance. Many methods may be employed to study specific environmental microbiology/ecology questions. Summary Microbes are a key component of environment and may contain valuable compounds/info. They are responsible for important geochemical cycling. Bacteria occupy various ecological “niches” based on their tolerance (or lack thereof) for salinities, temperatures, and baro-tolerance. Many methods may be employed to study specific environmental microbiology/ecology questions.

28. QUESTIONS?