Biology Department, University of Regina (Apr 4th, 2022)

Jesse McNichol (he/him) : PhD, Biological Oceanography

Postdoctoral Scholar, University of Southern California

Winogradsky Columns, or "The World in a Tube"

(Biology 310, Microbial Diversity and Cell Function)

Donato Giovanelli

Roadmap

  1. Group discussion activity
  2. Biogeochemistry of oxygen and carbon
  3. How to make a Winogradsky Column
  4. Chemistry of the microcosm
  5. Microbial metabolisms, niches
  6. Global implications
  7. Final activity on targeted isolation
  • Clicker questions / group discussions
  • Worksheet activity at end of class

Roadmap

  1. Group discussion activity
  2. Biogeochemistry of oxygen and carbon
  3. How to make a Winogradsky Column
  4. Chemistry of the microcosm
  5. Microbial metabolisms, niches
  6. Global implications
  7. Final activity on targeted isolation
  • Clicker questions / group discussions
  • Worksheet activity at end of class

Discuss in pairs for 2-3 minutes:

As part of a mission, you are sealed inside a spaceship for 1 year.

  1. What 4 basic things do you need to survive?
  2. Where do these 4 things come from on Earth?

Discussion

Discuss in pairs for 2-3 minutes:

As part of a mission, you are sealed inside a spaceship for 1 year.

  1. What 4 basic things do you need to survive?
  2. Where do these 4 things come from on Earth?

Discussion

  1. Water
  2. Food
  3. O2 source
  4. CO2 sink
  1. Rain
  2. Plants  
  3. Plants
  4. Plants

The carbon and oxygen cycle

Joseph Priestly's not-very-nice-to-mice experiment (bio.libretexts.org)

Everything that the plants take from the air they give to animals, the animals return it to the air; this is the eternal circle in which life revolves but where matter only changes place.’’                                  —Jean Baptiste Dumas, 1842

Consume food / O2 & produce CO2

Consume CO2 & produce food / O2

The carbon and oxygen cycle

Elemental cycles + geology + physics =

Earth Systems Science / Biogeochemistry

The carbon and oxygen cycle - Redox

Oxidize organic carbon to CO2 & reduce O2 to water

Reduce CO2 to organic matter & oxidize water to O2

Redox cycles of other elements

Falkowski, Fenchel and Delong, 2008

Interconnected redox cycles

Winogradsky columns

A microcosm of C, O, S redox cycling...

O2

H2S

Winogradsky: the 1st Microbial Ecologist

... helps us to understand global processes

One global system

Redox cycles of other elements

Winogradsky columns: a recipe

Microbiology à la Winogradsky:

Combine in a bottle or tube:

  1. Organic carbon source
  2. Inorganic carbon source
  3. Sulfur source
  4. Water (fresh or salty)
  5. Mud / sediment

    Mix well and incubate in the light. Combines well with research. Keeps for up to 20 years.

Winogradsky columns: a web of reactions

Think-pair-share (3 minutes):

  1. Give an example of the waste product of one organism feeding another
  2. What would happen if you sealed the cap and incubated in the dark?

Winogradsky columns: metabolic rationale

H2S

O2

Coupling of redox reactions

Coupling of redox reactions

Coupling of redox reactions

Electron & proton flow during respiration

Electron & proton flow during respiration

Electron & proton flow during respiration

Electron & proton flow during respiration

Electron & proton flow during respiration

Oxygen production occurs as a byproduct of oxidizing water

O2 production in a phototroph

Taiz and Zeiger, Plant Physiology, 4th edition

Electron transfer in phototrophs

Do oxygenic phototrophs respire?

D) 2 & 3

E) 1 & 3

C) 2

B) 1 & 2

A) All of the above

  1. They do not need to respire because they produce oxygen internally and use light to produce ATP
  2. They might need to respire at night or when light levels are too low
  3. Cells of plants that are not exposed to light like roots need to respire

Do oxygenic phototrophs respire?

D) 2 & 3

E) 1 & 3

C) 2

B) 1 & 2

A) All of the above

  1. They do not need to respire because they produce oxygen internally and use light to produce ATP
  2. They might need to respire at night or when light levels are too low
  3. Cells of plants that are not exposed to light like roots need to respire

Do oxygenic phototrophs respire?

The carbon and oxygen cycle in a eukaryotic alga

Chloroplast

Mitochondrion

The whole carbon cycle in a single cell!

Winogradsky columns: classifying metabolisms

H2S

O2

How to classify these groups metabolically?

Classifying metabolism

Classifying metabolism

Classifying metabolism

Classifying metabolism

Classifying metabolism

Classifying metabolism

Classifying metabolism

Classifying metabolism

Photo-litho-autotrophs

Chemo-organo-heterotrophs

Classifying metabolism: Oxygen

Diagram: Matthew Kirk

Classifying metabolism: Oxygen

Anaerobic

Aerobic

Classifying metabolism

Oxygenic photo-litho-autotrophs

Anaerobic Chemo-organo-heterotrophs

Anoxygenic photo-litho-autotrophs

Classifying metabolism

Think-pair-share (3 minutes) - Where would you find:

  1. Aerobic chemolithoautotrophs
  2. Anaerobic methane oxidizers

Oxygenic photo-litho-autotrophs

Anaerobic Chemo-organo-heterotrophs

Anoxygenic photo-litho-autotrophs

A window back in time

The deeper you go, the more ancient the metabolic pathways

A window back in time

Biomarkers for anoxygenic photolithoautotrophs in ancient sedimentary rocks indicate euxinia, i.e. H2S in the sunlit ocean.

  • Since H2S is highly toxic, possible factor in mass extinctions

Diagram: Alatleephillips

  • Which microbes catalyze specific bio(geo)chemical processes?
  • What are specific proteins / pathways responsible and their properties?

DNA sequencing is now low-cost...

...allowing us to sequence environmental "microbiomes"

Methods for "mapping" microbial communities

  • Metabarcoding uses PCR to measure abundance of microbial life across space / time
    • Comprehensive (universal primers)
    • Sensitive (sequencing is cheap!)
    • Specific ("denoising" algorithms)

Microbe art: @claudia_traboni

Methods for "mapping" microbial communities

  • Cultivation still critical for microbial ecology
    • Physiology
    • Morphology, other traits
  • Growing O2-sensitive microbes is pretty easy:

DIY gassing station

Hungate tubes

Methods for "mapping" microbial communities

FISH

SIP

Fluorescence in situ hybridization (FISH)

Methods for "mapping" microbial communities

Techniques for working at the microbial scale

Sebastián & Gasol (2019), 10.1098/rstb.2019.0083 ; McNichol et al (2018) 10.1073/pnas.1804351115 ; Zehr (2015) 10.1126/science.aac9752; Bramucci et al (2021) 10.1038/s43705-021-00079-z

Methods for "mapping" microbial communities

  • Taxonomy (CARD/HCR-FISH) / transcription (FISH-TAMB)
  • Respiration, cell viability (RSG); Polyphosphate content (DAPI)

Automatic cell-sorting + environmental databases = taxon-specific activity or genetic potential measurements

Many uses for sorted cells:

  • Taxon-specific isotope uptake
  • Mini-metagenomes with few cells
  • Cell isolation for cultivation

Pjevac et al (2019) 10.1111/1462-2920.14739

Multiple sorting axes now possible

Methods for "mapping" microbial communities

Activity

  • Design an experiment to cultivate an organism (2 minutes), presenting in groups of 4+ (5 minutes)
    • Present metabolic classification
    • Describe electron donors, acceptors
    • Other special conditions needed (light, oxygen, etc) & suggest an environmental source

Oxygenic photo-litho-autotrophs

Anaerobic Chemo-organo-heterotrophs

Anoxygenic photo-litho-autotrophs

Review: Learning objectives

  • Describe how to construct a Winogradsky column and what each added component contributes to the miniature microbial ecosystem
    • Compare and contrast the environmental niches of the following organisms using the 3-part metabolic classification scheme:
        A) Oxygenic photolithoautotrophs
        B) Purple / green sulfur bacteria (non-oxygenic photolithoautotrophs)
        C & D) Aerobic / Anaerobic chemoorganoheterotrophs
    • Explain how the metabolism of microbes leads to visible redox zonation across depth
    • Explain 2 or more products of microbial metabolism in sediments that have ecosystem- or biosphere-wide impacts (any chemical you like)
        • Suggest a strategy for culturing any of the organisms in the above categories (except for aerobic heterotrophs / oxygenic phototrophs) including carbon source, electron donors, and electron acceptors needed, and a possible environmental source (be creative!)

Closing thought

At the very core of Dubos’ scientific vision lay Vinogradskii’s perception that ‘‘countless microbes perform limited, well-defined tasks to recycle organic matter so that it does not accumulate in nature.’’" —Ackert 2007

Next time: Active research

Cheap DNA sequencing => insights into evolution, biogeochemistry:

Prokaryotes:

  • Novel lineages
  • Pangenomes

Eukaryotes:

  • Huge unsampled diversity
  • Non-coding DNA &  "function"