Red Layer Microbial ObservatoryBiology 507 - In-Lab Workshop

Photosynthetic Microbes from Local Rivers & Beyond

Schedule of Activities Session One

1. Microbial Diversity & the RLMO Program2. Photosynthetic Microbes3. Mud Enrichment Techniques4. Microscopic Diversity

75% grade = in-lab projects, Word templates25% grade = curriculum/application essay

Introduction

Microbial DiversityProkaryotes: Bacteria and ArchaebacteriaEukaryotes: Fungi, Algae, and Protozoa

Culture-Dependent IdentificationPlate, grow, isolate microbes using mediaIdentify isolates - media-based testsNaming traditionally requires “pure cultures”

These approaches grow only 1-5% of what is present naturally in any environment.

Culture-Independent IdentificationExtract/describe biological moleculese.g. DNA, proteins/enzymes, pigmentsApply of specific probes or antibodies (Ab)Assess environment for metabolic processes

This workshop combines both culture-dependent and -independent approaches.

NSF/MOEnhance awareness of microbial diversityDiscover new ways to describe new microbesRLMO - photosynthetic bacteria, Yellowstone

MO projects worldwide - soil, open ocean, polar, urban (homeland security issues)…

Photosynthetic Microbes

Algae - eukaryoticCyanobacteria - bacteriaPurple - bacteria/ProteobacteriaGreen Sulfur - bacteria, unique phylumGreen Nonsulfur - bacteria, unique phylumHeliobacteria - bacteria/Gram PositiveHalophiles - archaea… next week

Algae/Cyano. = oxygenic; others = anoxygenic

Overall Reaction - Oxygenic/Plant-LikeLight + pigment: H2O -> O2 + (H+) + (e-)ATP synthesis via e- transport chain (ETC)ATP used to fix CO2 (“dark reaction”)

Summary ReactionH2O + CO2 yields O2 + glucose + ATPWe say that this is “oxygenic” photosynthesis.

Oxygenic Membrane SystemsPlants/Algae - chloroplast with thylakoidCyanobacteria - simple stacks, no organelles

Will isolate/assess pigments next time.

Algae DiversityGreen - cellulose walls, fresh/marineEuglena - no wall, most freshDinoflagellates - cellulose walls, marine - toxinsGolden - silica walls, fresh/marine - toxinsBrown - cellulose walls, marineRed - cellulose walls, marine

Euglena, Dinoflagellates, Golden exist only in unicellular forms - no multicellular.

Cyanobacteria DiversityWidespread habitats - deserts to sea iceTypical bacterial walls - peptidoglycanUnicellular/Non-N2-Fixing - SynechococcusFilamentous/non-N2-Fixing - OscillatoriaFilamentous/N2-Fixing - Anabaena

Noteworthy products of some include earthygeosmins, bloom-associated neurotoxins.

Overall Reaction - Anoxygenic Bacteria*Light + pigment: H2S -> S + (H+) + (e-)ATP synthesis via e- transport chain (ETC)ATP used to fix CO2 (“dark reaction”)

Summary ReactionH2S + CO2 yields S + glucose + ATPSulfur tolerate/use HIGH sulfur. Nonsulfur low.

*Archaeal photosynthesis TOTALLY different.

Anoxygenic Membrane SystemsPurples - lamellae (simple stacks)Green - chlorosomes (vesicles)Heliobacteria - none, all on cell membrane

Purple Bacteria DiversityAnoxic lakes, springs, mud - some thermalHigh levels H2S (sulfur) or lower (nonsulfur)MANY shapes - cocci, rods, spirilla, ovals…e.g. genus - Rhodospirillum

Green Sulfur DiversityAnoxic sulfur lakes, sediments - few thermalMANY shapes - cocci, rods, ovals…e.g. genus - Chlorobium

Green Nonsulfur DiversityHot spring mats, marine salt marshesAll filamentous, with cells being large rodse.g. genus - Chloroflexus

Heliobacteria (Gram Positive) Diversity Anoxic lakes, sediments - few thermalClosest relatives: Bacillus and ClostridiumRods that, like relatives, form sporesAlso can ferment chemicals, like relatives

Mud Enrichment Techniques

Winogradsky Column1980s - mud mixed with C source, buffer…Packed into graduated cylinder placed in lightSustains oxygenics (top), anoxygenics (middle)

Sealed Bottles1 tsp mud plus known media (S or no S)Tightly corked and placed in light

Other Ways to Exclude OxygenCandle jars - just like they soundShake tubes - tricky to make, get cultures out ofCommercial anaerobic jars - expensive

Microscopy Techniques

Standard Wet MountAgents live, applied with liquid to slide/coverslipNo need for stain - agents pigments, largeAlgae, Cyanobacteria - use 20-40X objective

Fixed Slides/Simple StainAgents dead, dried and heated to just slideAgents extremely small, pigment lightOther prokaryotes - need oil/100X