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A report of independent projects by Juergen Breitung

Microbial Diversity

Summer Course 1991

A) Green phDtotrophic bacteria isolated from a Winograsky column

A Winogradsky column, named for the famous Russian Microbiologist Sergei

Winogradoky, was prepared by filling a large lass cylinder (2,000 ml)

about one fourth full with organic rich sulfide containing mud from the

Sippewisset marsh. The mud was spiked with calciumsulfate as sulfate

source. The mud was then covered with seawater till to the top and

sealed with parafilm. The column was placed at a north window so as to

receive adequate ( but not excessive ) sunlight and left to develop for

several weeks. A bloom of green sulfur bacteria was observed and

microscopical observation showed an abundance of Prosthecochloris

Samples were withrawn from the column and added to agar dilution shakes.

After one and a half week green colonies were picked out of the fourth

and fifth agar dilution tubes and transferred anaerobically to a liquid

medium described for phototrophic sulfur bacteria. Three pure cultures

of Frosthecochloris were obtained after a growth period of one week.

Literature:

Brock,T.D. and Madigan M.T. (1991) Biology of Microorganisms; Sixth

Edition; Prentice Ran. International; 614—617

Starr,M.P. et al.(1981) The Prokaryotes; Volume 1 ; 279—290

B) Carbonmonoxide oxidizing sulfate reducing bacteria

Introduction: From the literature it is known that some sulfate reducers

possess. a carbonmonoxid dehydrogenase and are therefore able to grow on

carbonmonoxide as energy source plus acetate as carbon source (1). In

order to enrich for Co oxidizers which are probably not described yet,

two strategies were employed

Materials and Methods: 1.) Direct enrichment

160 ml serum bottles were anaerobically (N2/C02) filled with 50 ml

marine sulfate reducer medium using Hungate technique. subsequently

approx. 3 g black Sippewisset marsh sediment were added and the bottles

were sealed with black mushroom stoppers. Two bottles serves as a

control, two had 2%, two had 5% and two had 20 % CO in the headspace.

The bottles were incubated for two weeks on a shaker (80 rpm) at 30 DC.

Result: No H2S production could be detected after two weeks using the

diagnostic reaction for estimation of formed sulfide handed out during

the course. Also no turbidity could be detected.

2.) The second enrichment took advantage of the sulfate reducer

enrichment took place in the course. Six sulfate reducer enrichments of

different course members on lactate were used (second or third

transfer). Lactate enriches for sulfate reducers which can oxidize this

compound only to acetate ( Desulfov.Lbrio, Desulfomonas etc.). For some

of these “non acetate oxidizers” CO consumption is described (1). Each

of these enrichments were inoculated (10%) into four hungate tubes: tube

A contains 10 ml marine sulfate reducer medium plus 5 mM acetate plus 1%

CO in the headspace (0.17 ml 100% CO/ 17 ml headspace); tube B contains

10 ml s.r. medium plus 5 mM acetate; tube C contains 10 ml s.r. medium

plus 1% CC in the headspace and tube D contains 10 ml s.r. medium

without substrate as a control.

Results After four days in tube A/s (from Ron) turbidity could be seenand a yellow brown color in the test indicating growth of a sulfate

reducer. In order to test if it is reducible, 1 ml of tube A/S were

inoculated in tube A,B,C and D. And again after four days of incubationat 30°C turbidity and H2S was only detected in tube A. Microscopical

observation showed that about 70% of the bacteria are vibrios similar

shaped as DesulfovLbrio desulfuricans. With a 16 srRNA fluorescence

probe (described by Gunter) the vibrios showed a positive reaction with

the sulfate reducer probe. From this enrichment an agar dilution shake

was made in order to get a pure culture of this bacterium. The agar

shakes were made with marine sulfate reducer medium plus S mM acetate

plus 1 % CO in the headspace. After five days of incubation at 30°C tiny

yellow brownish colonies were detected and subsequently transferred

anaerobically in liquid sulfate reducer medium with 5 mM acetate plus 1

% CO in the headspace. This tranfer was made three days before finishing

the course and since then no growth was detected.

Conlusion With a four tube test ( CO plus acetate; acetate; CO;

control)a sulfate reducer was highly enriched, which uses Co as electron

source and acetate as carbon source. Most of the organisms were vibrio

shaped and it is postulated that a DesulfovLbrio species carries out the

following:

Reaction 1: 4CC + 41120b 4 CO2 + 4112

—80 kJf mol CO

Reaction 2: 4 2 + 5042 + 2H ‘ H2S + 4 1120

6 —152 kJ/ mol

X.iterature: Lupton,F.S. et al. (1984) PENS Microbiology Letters 263—268

C) Methane sulfonic acide (MSA) oxidizing or reducing anaerobic bacteria

Introduction: NSA is regarded as a major biogenic organic sulfur

compound. It is generated via atmospheric oxidation of dimethyl sulfide

with photochemically produced OH radicals, Virtually nothing is known

about the anaerobic biological fate of NSA. It enters into chemical

combination with soil and water contituents when deposited in rain or

snow. Anaerobic degradation seems to be likely in natural environments.

Direct use by methanogens ( reduction of the methylgroup) and sulfatereducing bacteria ( reduction of the sulfite group and/or oxidation ofthe methyl group or reduction of the methylgroup with extern electrons)

might be possible.

Materials and Methods: In order to enrich for a NSA utilizing anaerobic

bacterium the following hungate tubes were anaerobically filled with

following compounds in 15 ml marine sulfate reducer medium without

sulfate. Sulfate was added as indicated.

1) 2 ml sample; 10 mM NSA

2) 2 ml sample; 20 mM NSA

3) 2 ml sample; 10 mM NSA; 2 mM acetate

4) 2 ml sample; 2 mM acetate

5) 2 ml sample; 10 triM NSA; 20 mM sulfate

6) 2 ml sample; 10 mM NSA; 20 mM sulfate; 2 mM acetate

7) 2 ml sample; 20 mM sulfate; 2 mM acetate

8) no sample; 10 mM NSA as a control

9) 2 ml sample; without substrate as a control

Three different samples were used: Sippewisset marsh mud; mud from the

above mentioned Winogradaky column; and mud from a little pond at the

end of the parking aerea at the bike path. The 27 tubes were incubated

under anaerobic conditions (N2/C02) at 30 C.

Results: After twelve days turbidity could be seen in some tubes.

Therefore the tubes were checked for M2S production and methane

production in the headspace. Tube 9 showed little fl2S and methane

production depending on the sample taken. So, little methanogenesis and

M2S production occurs without added MSA and/or acetate. Only tubes

showed significant R25 and methane production comparing with the control

tubes:tube 3 and 6 with the Winogradsky column sediment sample. H2S and

methane were produced approximately in equimolar amounts.

Conclusion: The decribed simultaneous H2S and methane production only

with MSA plus acetate results in the following working hypothesis:

Acetate is oxidized and the electrons are used to reduce NSA to methane

and 1125.

C R3 - COO 14

%FpcJ’ktitaj

L C

CH11 4.W1S

Prorsed 94&4tL%

4 CH3—SO3 + 3 CH3—COOH

_____

4 CR4 + 4 HS + 6 Co2 + 6 H20)

The reduction of the methyl group and of the sulfite group are exergonic

reactions. In the case of tube 7 sulfate has first to be activated and

growth was therefore slow.

Whether it is one organism which carries out the overall reaction or

whether it is a interspecies hydrogen transfer could not be resolved and

should be worked out in the future. The results shows that NSA is used

as electron acceptor and is reduced to H2S and methane rather than

beeing oxidized.

Literature:

Kelly, P.R. and Baker, S.C. (1990) FEKS Microbiology Reviews 87;241—246

D) Enrichment of an anaerobic methane oxidizing bacterium

Xntroduction:Anoxic sediments and digested sewage sludge anaerobically

oxidized methane to carbon dioxide. This strictly anaerobic process

showed a temperature optimum between 25 and 37 0C, indicating an active

microbial participation in this reaction. With nitrate or ferrous iron

as electron acceptor the methane oxidation to carbon dioxid is a

exergonic reaction. However, until now no organism was isolated which is

able to grow anaerobically on methane as energy and/or carbon source.

Materials and Methods: In order to isolate an anaerobic methane

oxidizing bacterium several samples were taken. 160 ml serumbottles were

anaerobically filled with 40 ml methanogens or sulfate reducer medium

(freshwatermedium without sulfate) and 10 mM KNO3 or 10 mM Fed3 as

electron donor were added. Furthermore 0.3 ml 3 % yeast extract was

added for unknown growth factors. About 2 ml mud samples from cedar

swamp and a little freshwater pond at the end of the parking aerea at

the bike path were filled to the medium. 99 % methane was given to the

headepace with an 1.5 bar overpressure. Appropriate controls ( without

or,

electron acceptors; or without samples) were made. All bottles were

incubated at 30 °C and shaked with 80 rpm.

Results: A decrease of methane, which was measured with a

gaschromatograph, could not be detected in any of the bottles.

Conlusion: The anaerobic methane oxidizing bacterium remained to be a

miracle, probably it does not exist because of the difficulty of the

first oxidation step to methanol.

Literature: Zehnder, A.J.S. and Erock, T.D. ( 1980) Environmental

Microbiology ;39; 194—204.