Bacteriology 102:
Enrichment and Isolation
of Purple Non-Sulfur
Photosynthetic Bacteria

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Purple Non-Sulfur
Photosynthetic Bacteria
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This page is relevant to the laboratory experiment on the enrichment and isolation of purple non-sulfur photosynthetic bacteria as it has been taught in the UW-Madison Microbiology 102 course, and it is not intended to be authoritative for applied or research purposes.

We are not qualified to address applications of these and similar bacteria to bioremediation or other environmental problems, although we are certainly happy to learn about such activities.

No implication that we actually stock and distribute pure cultures of these organisms can be inferred. On this page we show how to isolate your own. For certified cultures for research or industry, please go to suppliers of such cultures – for example, ARS/NRRL and ATCC.

Text and photos copyrighted by John Lindquist. When emailing me, please cite the URL of this page. Copies of this page found elsewhere are not authorized and may contain spurious items.


I. Introduction to the Purple Non-Sulfur Photosynthetic Bacteria.

The Purple Non-Sulfur Photosynthetic Bacteria constitute a non-taxonomic group of versatile organisms in which most can grow as photoheterotrophs, photoautotrophs or chemoheterotrophs – switching from one mode to another depending on conditions available, especially the following: degree of anaerobiosis, availability of carbon source (CO2 for autotrophic growth, organic compounds for heterotrophic growth), and availability of light (needed for phototrophic growth).

Originally it was thought that these bacteria could not use hydrogen sulfide as an electron donor for the reduction of carbon dioxide when growing photoautotrophically, hence the use of "non-sulfur" in their group name. Sulfide can be used if present in a low concentration. Higher concentrations of H2S (in which the purple and green sulfur bacteria can thrive) are toxic, however. There is more about sulfide and sources of reducing power in section II, below.

Chemotrophic growth for the purple non-sulfur bacteria is achieved by respiration, although there are some exceptional strains and species which can obtain energy by fermentation or anaerobic respiration.

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xHere is an older approach to the phylogenetic grouping of the photosynthetic bacteria. The following summary of photosynthetic bacteria appeared in the Bacteriology 102 lab manual prior to the 2nd printing of the 2nd edition in the mid-1990s (General Microbiology: A Laboratory Manual by J. A. Lindquist (ed.), McGraw-Hill/Primis Custom Publishing – retired at its fourth edition). The anoxygenic and oxygenic groupings correspond to Sections 18 and 19, respectively, of Bergey's Manual of Systematic Bacteriology (First Ed., Vol. 3, 1989).

group of bacteria chlorophylls electron donor for photoautotrophy photoheterotrophy? chemotrophy?
Anoxygenic Photosynthetic Bacteria
Purple Sulfur Bacteria of the Family Chromatiaceae bchl a or b S or So or H2 (So globules formed inside cell from S) some spp. some spp.
Purple Sulfur Bacteria of the Family Ectothiorhodospiraceae bchl a or b S or H2 (So globules formed outside cell from S) possibly all spp. some spp.
xPurple Non-Sulfur Bacteria (Family Rhodospirillaceae*) bchl a or b Prob. all: H2 . Some: low levels of S, S2O3, So all spp. probably all spp.
Green Sulfur Bacteria (including Family Chlorobiaceae*) mainly bchl c, d or e S or So (So globules formed outside cell from S) potentially all spp. none
Multicellular Filamentous Green Bacteria (including Family Chloroflexaceae*) one or more of bchl a, c, d ? (photoautotrophy?) all spp. probably all spp.
Oxygenic Photosynthetic Bacteria
Cyanobacteria chl a H2O some spp.? some spp.
Prochlorophytes chl a and b H2O ? prob. none

* No longer recognized as a discrete taxonomic group according to Bergey's Manual of Systematic Bacteriology (First Ed., Vol. 3, 1989).


xAn updated taxonomic arrangement according to The Prokaryotes and the 2nd Edition of Bergey's Manual of Systematic Bacteriology may be added here. In these resources, the inclusion of purple non-sulfur photosynthetic bacteria in the Alphaproteobacteria and the Betaproteobacteria is discussed.


II. A Few Words about Photosynthesis and the Source of Reducing Power.

The traditional "photosynthetic equation" many of us grew up with is as follows:
xCO2 + H2O = CH2O + O2
This equation is meant to represent the incorporation of carbon dioxide into cellular carbohydrates with the utilization of water as the source of reducing power (electron donor) and the consequent release of oxygen. This equation is more accurately given as follows:
xCO2 + 2H2O = CH2O + H2O + O2
The cyanobacteria are oxygenic (oxygen-producing) photosynthetic bacteria that possess this type of photosynthesis along with plants and algae. The use of carbon dioxide as the carbon source and an inorganic compound (water) as the source of reducing power is reflected in the terms autotroph and lithotroph (respectively) which are used for an organism performing this reaction. With light as the ultimate source of energy, the organism would also be termed a phototroph.

The preceding equation can be made more general by substituting "A" for the oxygen ("O") in the source of reducing power when it is realized that compounds other than water can serve in that capacity for certain other types of organisms:
xCO2 + 2H2A = CH2O + H2O + 2A
For the purple and green sulfur bacteria, hydrogen sulfide is utilized, and the equation is given thus:
xCO2 + 2H2S = CH2O + H2O + 2S

Note the column in the table above which lists electron donors used for (photo)autotrophic growth by the various groups of organisms. For those organisms which can grow as heterotrophs – such as the purple non-sulfur photosynthetic bacteria – we would expect that various organic compounds could serve as electron donors. New editions of Bergey's Manual and modern textbooks (such as Brock) can be consulted for further information. There is more about energy generation and reducing power here.


III. Enrichment and Isolation of Purple Non-Sulfur Photosynthetic Bacteria.

In looking for the purple non-sulfur bacteria, we find it most advantageous to set up conditions for photoheterotrophic growth, utilizing a source of light, anaerobic conditions (needed for phototrophic growth by these organisms), no hydrogen sulfide, and an organic carbon source not generally used by other bacteria under these conditions such as sodium succinate or malate. Note the medium formulas below. Not only will most other types of organisms be restricted from growing, but the purple non-sulfur photosynthetic bacteria will be easily recognized by the presence of photosynthetic pigments. When substantial pigmented growth shows up in the liquid medium or is seen in the natural source, it is referred to as a "bloom."

One may expect these organisms in their most likely habitat – i.e., anaerobic mud in ponds and lakes where there is access to sunlight. Other successful sources where they can be found as easily-recoverable contaminants include surface water from streams, bogs and transient puddles – and even rain, snow, icicles and hailstones! High concentrations of these organisms have even been found in the water trapped by the leaves of bromeliads and pitcher plants. Soil and flat leaf surfaces are worth a try.

Isolation of purple non-sulfur bacteria is accomplished easily by adding the source material to a liquid enrichment medium in a stoppered bottle. The final volume attained can be approximately 5-25% inoculum (a lower amount if a solid sample is used) with the medium filling up the rest of the container such that no air bubbles are trapped. These enrichments are placed near a tungsten light (one or more common desk lamps) at room temperature to 30°C. Once the enrichment has achieved turbidity with the consequent bloom, it is streaked onto plates which are then incubated anaerobically near the light source.

Direct inoculation of source material (by loop-streaking or application of 0.1 to 1 ml) onto plates may result in very few (if any) colonies of these organisms showing up. If 5-100 ml (or more) of the sample is run through a 0.45 micrometer filter – followed by placing the filter (organism side up) on the medium – one can achieve substantial numbers and varieties of colonies.

x

THE ABOVE LEFT PHOTO shows enrichments in Mineral Salts-Succinate Broth (medium formula below) showing the characteristic "bloom" after about a week of incubation in the light at 30°C. So here is a good question: After filling the bottles completely with sample and medium, how are anaerobic conditions actually achieved? Hint: What bacterial process (associated with energy generation) is responsible for "using up" the oxygen? (The large bottle on the right is not filled to the top but it has a layer of mineral oil floating on the medium. The same answer to the above question will apply here as well.)

THE ABOVE RIGHT PHOTO shows a plate streaked from an enrichment and then incubated anaerobically in the light. Note the pigmented colonies of purple non-sulfur photosynthetic bacteria.

x

THE PHOTO ON THE RIGHT is a closeup of a 0.45 micrometer filter showing a variety of different colony types growing upon it after about a week's incubation. Originally, a 50 ml water sample was passed through the filter, and then the filter was placed on the solid medium in a petri dish followed by incubation in the light under anaerobic conditions at 30°C. (This plate is actually overcrowded, and a 5 ml sample would have worked better.)

One must appreciate the fact that the purple non-sulfur bacteria would probably be overrun (crowded out) by various respiring chemotrophs from the sample if our enrichments and plates were incubated under aerobic conditions. Aerobically, the photosynthetic pigments would not be as visible (if at all), and picking likely colonies of these organisms would be difficult or impossible. Likewise, they would probably be overrun by fermenting chemotrophs if our enrichments and plates were incubated under anaerobic conditions with a "popular" carbon source such as glucose in the medium.

An example of how our procedure can be summarized on a flow chart is seen here.


IV. Formulas for the Mineral Salts-Succinate Broth and Agar Media.

Of the several we have used in teaching and research over the decades, the medium detailed below has probably given us the best luck in obtaining rapid and substantial growth (enrichments and colonies) of purple non-sulfur photosynthetic bacteria. It is probably based on a medium formulated originally by Norbert Pfennig who was a distinguished and well-remembered visitor to our department.

With water samples containing a substantial amount of oxygenic phototrophs (mainly cyanobacteria and green algae), it may be possible to have these organisms come up within a week and overtake the enrichments. An example of this happening is shown here. Therefore, it is probably best to streak your plates from the enrichments as soon as a reddish "bloom" of the desired organisms is evident.

BASAL MEDIUM  
  KH2PO4 0.33 g  
  MgSO4.7H2O 0.33 g  
  NaCl 0.33 g  TRACE SALTS SOLUTION: 
  NH4Cl 0.5 g   ZnSO4.7H2O 10 mg
  CaCl2.2H2O 0.05 g   MnCl2.4H2O 3 mg
  Sodium succinate 1.0 g   H3BO3 30 mg
  Yeast extract 0.02 g   CoCl2.6H2O 20 mg
  Distilled H2O 1.0 L   CuCl2.2H2O 1 mg
  pH 6.8-7.2   NiCl2.6H2O 2 mg
Sterile solutions added after autoclaving the above:   Na2MoO4 3 mg
  Trace salts solution (see at right) 1.0 ml   Distilled H2O 1.0 L
  0.02% FeSO4.7H2O solution 0.5 ml   pH 3-4
  • For a solid medium, add 15.0 g agar per liter of Basal Medium.
  • The above formulation works well when one is enriching or isolating directly from natural sources and minimal interference from other organisms is especially desirable.
  • For maintenance of pure cultures or when streaking plates from enrichments, add another 1.0 g of sodium succinate and 1.0 g of yeast extract per liter of Basal Medium. These items encourage robust growth of the desired organisms and also make detection and avoidance of undesired colonies easier.

V. Characterization of Isolates.

Among the tests and observations we perform on our individual isolates in Bacteriology 102, we determine cellular morphology. The following can be summarized about the four genera we most frequently isolate: Rod-shaped cells would be either Rhodopseudomonas (if curved, "knobby" rods – reproducing by budding) or Rhodobacter (if straight rods or ovals – reproducing by binary fission). Oval to rod-shaped cells connected by thin filaments would be Rhodomicrobium, and spiral-shaped cells (the least-often isolated) would be Rhodospirillum.

x

We also do a test that is somewhat similar in its setup (but not so much in theory) to the oxygen relationship test which utilizes Thioglycollate Medium. In Thioglycollate Medium, it is important to recall that anaerobic growth is due only to fermentation. However, in the particular test considered here for the purple non-sulfur photosynthetic bacteria, anaerobic growth is only associated with anoxygenic phototrophy, and the categories associated with the standard oxygen relationship test (strict aerobe, facultative anaerobe, etc.) do not apply.

The test medium is the Mineral Salts-Succinate Agar with the extra succinate and yeast extract (see above). Tubes of the melted medium are inoculated in duplicate with an isolate such that the inoculum is dispersed evenly throughout the medium. The tubes are incubated at 30°C: one in the light and one in the dark. The following points should be noted when we characterize this isolate not as a facultative anaerobe (which implies fermentation) but rather as a facultative phototroph:

  • Typical of the anoxygenic phototrophic bacteria, the light-associated anaerobic growth is pigmented – a net color resulting from the photosynthetic and associated pigments. We have already noted such pigmentation in our enrichments and colonies.
  • Without anything in this medium to help with fermentation and anaerobic respiration, we expect (and get) no anaerobic growth in the dark.
  • Aerobic growth is due to aerobic respiration, a chemotrophic process. This growth tends to be less- or non-pigmented, and both tubes in the photo show such growth at the top of the medium.

How the results for individual isolates obtained in this experiment can be summarized in a table can be seen in the hypothetical example given here. We could do additional testing according to what is suggested in Bergey's Manual, in which case we could determine what species we have isolated.


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John Lindquist, Department of Bacteriology,
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