Post on 08-Apr-2018
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