FINAL ENRICHMENT PROJECTBy Matthew Laird ‘15

Presentation Outline

I. IntroductionII. The Organisms

I. MethylobacteriumI. PhysiologyII. HistoryIII. HabitatIV. Ecological ImportanceV. ApplicationsVI. Medium VII. The InoculumVIII. Incubation ConditionsIX. ResultsX. Interpretation of Results

Presentation Outline (continued)

II. Agar Decomposing Bacteria (Alteromonas)

I. PhysiologyII. MediumIII. InoculumIV. Incubation ConditionsV. ResultsVI. Interpretation of Results

III. Summary

Introduction• For my final enrichment project, I

decided to isolate two microbial organisms, one being Methylobacterium, because I was interested in its physiology. The second was sea agar decomposing bacteria (Alteromonas), which I chose because of the close proximity of my home to ocean water.

Methylobacterium

Physiology• The genus Methylobacterium is

composed of a variety of pink-pigmented facultatively methylotrophic (PPFM) bacteria.

• They are capable of growing on one carbon compounds such as formate, formaldehyde, and especially methanol as a sole carbon source.

Physiology (cont.)

• Members of Methylobacterium are strict aerobes and gram-negative, though sometime gram-variable.

• Are mainly rods, occasionally branched and exhibit polar growth.

• All strains are motile by a single sub polar/lateral flagellum.

• Contain citrate synthase.

Physiology (cont.)

• Their characteristic pink pigment is nondiffusible, nonfluorescent, and most likely a carotenoid.

• Are catalase and oxidase positive.• Have a complete tri-carboxylic acid

cycle when grown on complex organic substrates.

Physiology (cont.)

• Urease is produced by all strains.• All strains are Indole negative and TSI

negative (do not produce H2S). • Usually Nitrate-Reduction negative,

though some strains are positive.• The fatty-acid composition of PPF

cells is largely composed of mono-unsaturated acid chains.

Physiology (cont.)

• Sensitive to antibacterial agents: kanamycin, gentamycin, albamycin, streptomycin, framycetin, and especially tetracyclines.

• Resistant to antibacterial agents: cephalothin, nalidixic acid, penicillin, bacitracin, carbenicillin, colistin sulfate, polymixin B, and nitrofurantoin.

History• The first strain of Methylobacterium

was discovered in 1913 by Polish microbiologist Kazimierz Bassalik.

• This strain was isolated from a piece of an earthworm.

• However, the genus was not studied excessively until the 60’s/70’s when interest in its one-carbon assimilation pathways peaked.

Bassalik circa 1928

History (cont.)• There presently exists twenty-two validated

species of the genus Methylobacterium. • They are: Methylobacterium adhaesivum; M.

aminovorans; M. aquaticum; M. chloromethanicum; M. dichloromethanicum; M. extorquens; M. fujisawaense; M. hispanicum; M. isbiliense; M. lusitanum; M. mesophilicum; M. nodulans; M. organophilum; M. podarium; M. populi; M. radiotolerans; M. rhodesianum; M. rhodinum; M. suomiense; M. thiocyanatum; M. variabile; M. zatmanii.

Habitat• Members of the genus

Methylobacterium are ubiquitous and are thus found in a variety of habitats.

• Habitats include freshwater, lake sediments, leaf surfaces, rice grains, “air”, hospital environments, and in pharmaceutical preparations such as face creams.

Habitat (cont.)• Since members of Methylobacterium

are strict aerobes, they can be isolated from almost any freshwater source where dissolved oxygen is present.

• One species, M. organophilum, a methane-oxidizer, can only be found during the summer months in the upper stratified layers of lake, where methane is present.

Ecological Importance

• Methylobacterium may play an important ecological in the carbon cycle in nature.

• Methylobacterium strains have been localized as endosymbionts within cells in the buds of Scotch pine (Pinus sylvestris).

Ecological Importance

(cont.)• Methylobacterium forms a strong cohesive mat in fuel/water interfaces, such as those that t occur in storage tanks for middle distillate fuel-oils for heating or diesel engines. These chemofilms promote biofilm formation, which present potential problems for filters that may lead to engine failure.

Applications• Methylobacterium have the potential

to produce single-celled proteins from methanol, however, their bioconversion ratios do not stack up to those of other methylotrophs.

• Because of this, no immediate uses of this genus in this way is seen in the immediate future.

Applications (cont.)

• The pink carotenoid pigment in Methylobacterium strains has been used commercially as a dye as well as a colorant in food.

• It appears that Methylobacterium has some sort of connection with vehicular emissions, showing that these organisms are able to grow on the polycyclic aromatic hydrocarbons found in exhaust emissions.

• This suggests that these organisms might be used in the future as biological monitors of vehicle pollution.

The Medium• Because of the ability of

Methylobacterium to grow on methanol as sole carbon and energy source, can use methanol mineral salt medium (MMS) to isolate bacteria.

• Both MMS plates and broth can be and were made.

The Medium (cont.)

• Methanol Mineral Salts Medium• Chemicals:– The following are added perliter (L):(1-2% of sterileMethanol is added to mediumafter autoclaving.)

Ingredient: Amount:

K2HPO2 1.20 g

KH2PO2 0.62 g

CaCl2●6H20 0.05 g

MgSO4●7H2 0.20 g

NaCl 0.10 g

FeCl3●6H20 1.0 mg

(NH2)2SO4 0.5 ɥg

CuS04●5H20 5.0 ɥg

MnSO4●5H20 10.0 ɥg

Na2MOO4●2H20 10.0 ɥg

H3BO4 10.0 ɥg

ZnSO4●7H20 70.0 ɥg

CoCl2●6H20 5.0 ɥg

The Medium (cont.)

• Because chemicals in blue are added in such small amounts (ɥg/L), a stock solution of 100 mL was made.

• This stock solution was then added to the 1L medium solution to obtain the correct concentration of each chemical.

Why This Medium Is Selective• Looking at the chemicals in the MMS

media, the only carbon source is the methanol added after autoclaving.

• Thus, only methylotrophs, those organisms which can utilize methanol as a sole carbon source should be able to grow on the medium.

The Inoculum• Inoculum:• I previously stated that

Methylobacterium can usually be isolated from almost any freshwater source with dissolved oxygen.

• Thus, my inoculum was collected from Middle River, a fresh body of water here in Worcester.

Inoculum Video

Incubation Conditions

• MMS plates were streaked for isolation with the inoculum and then placed in an incubator at 30°C, optimal growth temperature.

• MMS tubes were placed in the shaker with caps loosely tightened to aerate the medium.

Results• Plates:

Results (cont.)• Broth:

Results (cont.)• Gram Stain:

Results (cont.)• Fermentation Tests:• Results: negative for Glucose, Lactose, and Sucrose.• This is expected, sinceMethylobacterium is anobligate aerobe.

G L S

Results (cont.)• TSI Test:• Previously, I stated that Methylobacterium strainswere TSI negative (not producingH2S).• Result: not negative, but alsono H2S either, since no blackprecipitate.

Results (cont.)• Indole Tests:• Previously, I stated that Methylobacterium strains were all Indole negative.• Result: negative test, consistent with literature.

Results (cont.)• Nitrate Reduction Test:• Previously, I stated that

Methylobacterium strains are usually Nitrate-Reduction negative, though some strains are positive.

• Result: positive for nitrate reduction, so consistent with literature.

Results (cont.)• Citrate Test:• Previously, I stated that all

Methylobacterium strains contained the enzyme citrate synthase.

• Result: positive citrate test, consistent with literature.

Results (cont.)• Urease Test:• Previously, I stated that all

Methylobacterium strains were urease positive. • Result: positive urease test, consistent with literature.

Results (cont.)• Catalase Test:• Previously, I stated that

Methylobacterium strains are catalase positive.

• Result: negative test, not consistent with literature.

Results (cont.)• Oxidase Test:• Previously, I stated that

Methylobacterium strains are oxidase positive.

• Result: negative test, not consistent with literature.

Interpretation of Results

• Many of the physiology tests matched that of the literature, while others did not or were ambiguous.

• The fact that the colonies were not their characteristic pink color leads me to believe that I did not isolate a member of the genus of Methylobacterium, but rather another methylotroph with a similar physiology.

Agar Decomposing

Bacteria (Alteromonas)

Physiology• The genus Alteromonas:• Aerobic, Gram-negative, non-

fermentative, heterotrophic, motile, non-pigmented, Pseudomonas-like bacteria able to decompose algal polysaccharides such as agar.

The Medium• Because of the ability of these

organisms to depose agar, sea water agar (SWAGAR) plates were made to isolate bacteria.

• The following are added per liter (L).

Ingredient: Amount:

Sea Water 500 mL

Agar 15 mLDistilled H20 500 mL

The Inoculum• Inoculum: • Inoculum was

sea water taken from Craigville Beach on Cape Cod in a town called Centerville, near where I live.

Results• Plates:

Results (cont.)• Fermentation: glucose, lactose,

sucrose all negative.• TSI: yellow/yellow. No H2S precipitate. • Indole: negative test.• Nitrate Reduction: negative for nitrate

reduction and nitrite reduction.• Citrate: negative• Urease: negative

Interpretation of Results

• The characteristic depressions that were found in the plates leads me to believe that I have in fact isolated a member of the genus Alteromonas.

• The physiology tests also go to confirm this belief.

Summary• The organism that I isolated in the

first part is likely not a member of the genus Methylobacterium, but rather another methylotroph will similar physiology.

• The organism that I isolated in the second part is very likely to be a member of the genus Alteromonas.

THANKYOU