Red Layer Microbial ObservatoryBiology 507 - In-Lab Workshop

Photosynthetic Microbes from Local Rivers & Beyond

Schedule of Activities Session Two

1. Photosynthesis, Pigments, Archaea2. Pigment Analysis Set-Up3. Follow-Up - Last Week4. Curriculum Discussion/Essay

5. We may or may not have time for each person to use the fluorescence microscope.

Oxygenic Anoxygenic

What is split? H2O H2S/H2

OrganismsPlants/Algae

Cyanobacteria

Purple BacteriaGreen BacteriaHeliobacteria

Major PigmentsChlorophylls

(shorter wavelenth)Bacteriochlorophylls(longer wavelengths)

Photosynthetic Pigments

LocationSpecific proteins hold in membrane - nonpolarSpecies-specific proteins define real absorptionNaked pigment uses discrete value - e.g. 770Species-specific proteins alter - e.g. 800, 900Different methods for naked vs. protein-bound

Chlorophylls (Chl) - OxygenicPlants - green a and b Algae - green a/b, gold/brown a/c, red a/dCyanobacteria - green a only

All of these, when excited by UV, “fluoresce” or emit light in the red or yellow range.

Accessory Pigments - OxygenicCarotenes - yellow or red, WIDESPREADXanthophylls (brown), phycoerythrin (red)

Bacteriochlorophylls (Bchl) - Anoxygenic Purple bacteria - Bchl aGreen bacteria - Bchl cHeliobacteria - Bchl g

Accessory Pigments - AnoxygenicCarotenes - yellow or red, WIDESPREADPhycocyanin - blue, similar to phycoerythrin

Many chemotrophic pathogens have carotenes because these neutralize H2O2.

Isolating Naked PigmentsBecause nonpolar, organic solvents involvedMethanol for research-grade work - GLOVESAcetone acceptable/available - harshBecause cell walls, you will pulverize sample Petroleum ether (jar/hood) - chromatographyData useful for major grouping assignments

A spectrophotometer allows one to skip chromatography.

Isolating Protein/Pigments IN MembraneDisrupt cells using just pulverization - expensiveFragmented pigments called “in vivo”Data useful for grouping and species variation

Archaeal “Photosynthesis” - Class By ItselfRemarkably, these are commercially available.

Why Unique?Bacteriorhodopsin (Brho) pigment in membraneLight causes Brho to physically move H+ outThis generates proton motive force - ATPaseNO ETC involved in this process

Brho homologous to eukaryotic rhodopsin.

Halophiles5.5+ M salt - many different ions and pHEvaporation ponds, salt lakes, salted foodsHalobacterium = phototropic halophile

Many other Halophiles are not photosynthetic. They are simple chemotrophs.

Photosynthesis and Evolution

Hot earth and the purple origin theoryAncient earth - 100°C but darkHeat gives off infrared - 900 nm or moreEmerging purple Bchl absorb this

Human eyes and salty Archaea - speculate all you want.

What does the tree of life support?Green nonsulfur most ancient - purple membersPhototrophy evolved many timesChloroplast most similar to Cyanobacteria

Pigment Analysis Set-Up

OverviewEach team will set up 2-3 different phototrophsYou will pulverize the sample in methanolExtract 15 minutes - ice/darkThen streak on chromatography paper, developAnd analyze the rest on a spectrophotometerShare class data - need all for web analysis

Analyze fluorescence data on web template.