Download Helen`s Project4

Bacteria in the BBB Ponds
In this project, we plan to investigate the bacteria in the BBB ponds and differences
in their photosynthetic light-harvesting centers.
Generally, light harvesting centers and their satellite complexes are composed of
bacteriochlorophylls and carotenes. We will identify these pigments in several different
cultures, including in purple sulfur bacteria (Chromatium spp) and green sulfur bacteria
(Chlorobium spp) using characteristic absorbance values and thin layer chromatography.
Comparing the Caltech strains with different pigments may allow us to estimate and
understand differences in energy yield.
Along the way, we will also be using mixed and single culture methods to isolate
bacteria and investigating the plate count anomaly using a variety of media. “Species” on
each plate will be categorized based on gross colony morphology and compared to plates of
other media and to the Winogradsky column, a mixed culture method.
Timeline:
Jan 28
Feb 2
Feb 4
Feb 9
Feb 11
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Feb 16
Feb 18
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Feb 23
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Feb 25
Mar 2
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Mar 4
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Mar 9
Mar 11
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Pond field trip- talk/discuss
Make Winogradsky column- mixed culture
Start plate count anomaly (mixed culture pure culture)
Retreat (Background reading)
Retreat (Background reading)
Select colorful colonies for streaking
Grow purple sulfur bacteria (ATCC Medium 37, infrared
lights?) Use Winogradsky for inoculum
Examine specimens from pure streaked culture with
microscope
Plate duplication:
o Broth or LB pond, minimal, min+carrot
Compare later with survival on new media.
Biodiversity calc
Scan absorbances of Bacteria--??
o like Overmann Paper- compare with his.
Use purple cultures and broth cultures on TLC to separate
pigments; try to identify carotenoids, etc…
Progress Report due
Bulk doubling time expts
Look at bacteria from Winogradsky column with
microscope
o Analyze: Same as from plate count anomaly?
Discuss QM of photosynthesis; estimate efficiencies of
strains with different pigments based on doubling time
Work on Presentations, practice talk
Presentations
Materials:
Winogradsky column: Mixed culture in a clear column or jar. (See “A Field Guide to
Bacteria” by Betsey Dexter Dyer.)
Pure culture: Growing a single strain of bacteria in liquid or on agar plates.
ATCC Medium 37 (for Chromatium species): See
http://www.atcc.org/Attachments/3682.pdf This includes vitamin B12, which is
necessary for rapid growth in Chromatium.
For plates, add 15 g agar per L of media. Autoclave, let cool. Add 1 mL Vit B 12 solution per
liter, mix and pour.
Chlorobium Media: (1 L)
0.30 g KH2PO4
0.34 g NH4Cl
0.34 g KCl
0.15 g CaCl2*2H2O
0.50 g MgSO4*7H2O
1.00 mL Trace solution
Autoclave, cool, and add:
15.0 ml 10% (w/v) NaHCO3 (saturated with CO2)
6.0 ml 10% (w/v) Na2S*9H2O (autoclaved)
1.0 ml Vitamin B12 solution (2 mg B12 in 100 ml distilled water)
Adjust pH to 6.7 with sterile 2 M H2SO4.
Dispense into 100-ml bottles.
Trace solution (1-liter)
2.00 g FeSO4*7H2O – dissolve in 10 ml HCl (25% w/v); add DI water and:
190 mg CoCl2*6H2O
100 mg MnCl2*4H2O
70 mg ZnCl2
36 mg Na2MoO4*2H2O
24 mg NiCl2*6H2O
6 mg H3BO3
2 mg CuCl2*2H2O
For plates, add 15 g agar per L of media. Autoclave, let cool. Add 1mL Vit B12 solution mix
and pour.
Minimal Media: To make M9 Salts aliquot 800ml H2O and add:
64g Na2HPO4-7H2O
15g KH2PO4
2.5g NaCl
5.0g NH4Cl
Stir until dissolved
Adjust to 1000ml with distilled H2O
Sterilize by autoclaving
Measure ~700ml of distilled H2O (sterile) and add 200ml of M9 salts, 2ml of 1M MgSO4
(sterile), 20 ml of 20% glucose (or other carbon source), 100ul of 1M CaCl2 (sterile) and
adjust to 1000ml with distilled H2O.
Pond Water Media: Filter sterilize 500 mL pond water. (add 15 g agar per L for plates,
autoclave and pour.)
Chicken/Beef Bouillon Media:
Dissolve 1 11g cube (Knorr) in 1L DDH2O. Autoclave. Skim off fat, and autoclave
again. For plates, add 15 g agar per L before second autoclaving.
(Alt.) Dissolve 1 cube in 2 L DDH2O. (Clearer, easier to see.)
Bibliography:
Andersson et al. Femtosecond dynamics of carotenoid-to-bacteriochlorophyll a energy
transfer in the light-harvesting antenna complexes from the purple bacterium Chromatium
purpuratum. Chemical Physics (1995) vol. 210 pp. 195-217
Delves into QM of photosynthesis.
Damjanovic et al. Energy transfer between carotenoids and bacteriochlorophylls in lightharvesting complex II of purple bacteria. (1999) vol. 59 (3) pp. 3293-3311
Explains pretty well how the light harvesting centers are organized and explains
electron transfer.
Devereux et al. Natural Relationships among Sulfate-Reducing Eubacteria. Journal of
Bacteriology (1989) pp. 1-7
16s sequences used to compare Desulfos.
Franz et al. Utilization of 'elemental' sulfur by different phototrophic sulfur bacteria
(Chromatiaceae, Ectothiorhodospiraceae): A sulfur K-edge XANES spectroscopy study. J.
Phys.: Conf. Ser. (2009) vol. 190 pp. 012200
Structure of carotenoids and what we can learn through structural studies.
Fujii et al. 1 H NMR, electronic-absorption and resonance-Raman spectra of isomeric
okenone as compared with those of isomeric i -carotene, canthaxanthin, i -apo-8'-carotenal
and spheroidene. Spectrochimica Acta, Part A (1998) vol. 54 pp. 727-743
More Carotene Structure
Imhoff. The Chromatiaceae. Prokaryotes (2006) (6) pp. 846-873
A good background on Chromatia spp., the purple sulfur bacteria. This is an excellent
place to start.
Leadbetter. Cultivation of recalcitrant microbes: cells are alive, well and revealing their
secrets in the 21st century laboratory. Current Opinion in Microbiology (2003) vol. 6 pp.
274-281
Background on how we can use selective enrichment to grow bacteria we’re looking
for.
Overmann and Glaeser. Selective enrichment and characterization of Roseospirillum
parvum, gen. nov. and sp. nov., a new purple non-sulfur bacterium with unusual light
absorption properties. Arch Microbiol (1999) vol. 171 pp. 405-416
Selective enrichment with light instead of media.
Reinhartz et al. Sulfide oxidation in the phototrophic sulfur bacterium Chromatium
vinosum. Arch Microbiol (1998) vol. 170 pp. 59-68
How Chromatium reduce sulfur.
Toropygina et al. Reconstitution of Okenone into Light Harvesting Complexes from
Allochromatium minutissium. Biochemistry (Moscow) (2005) vol. 70 (11) pp. 1231-1237
Some groups use Chromatium as a model system for bacterial photosynthesis. Here
they trade the normal carotene for high-yield okenone.
Whitman et al. Prokaryotes: The unseen majority. PNAS (1998) pp. 6578-6583
A neat discussion of the scale of prokaryote diversity and population.
Zengler et al. Cultivating the uncultured. PNAS (2002) vol. 99 (24) pp. 15681-15686
One approach to getting around the plate count anomaly with microfluidics.