Discussion

Evaluation:
We were successful in proving our hypothesis that Rhizobium can be isolated from root nodules of soybean and clover and grown on yeast mannitol agar plates. The enrichment method proved to be successful. We got the bacteria by crushing the nodules found on soybean and clover roots. We first wanted to use the Legume Extract Agar to cultivate Rhizobium. This medium required alfalfa roots, soybean meal, glucose, and various kinds of salts to help Rhizobium grow. Since it was hard to get alfalfa roots, we did not use this medium. Instead we prepared Liquid Nitrate Medium to be used as the medium to isolate Rhizobium. The presence of calcium pantothenate limits the growth of a few of the Rhizobium species. Since we were not sure which species would be present in clovers and did not want to limit ourselves, we decided against using this medium. Finally we decided to use Yeast Mannitol Agar medium to grow Rhizobium. Because this medium contains a source of nitrogen (in yeast extract) and carbohydrate (mannitol), it supports the growth of Rhizobium. The bacteria obtained from the root nodules were plated on YMA plates, and streaked over and over again until a pure colony was seen growing on the plate.

On the majority of the plates, white, mucilaginous, raised, semi-translucent colonies were seen. This fits the Bergey's manual description of genus Rhizobium as forming "copious extracellular polysaccharide slime." The clover plates, however, had slimy, yellow colored colonies forming on them. This morphological difference found in clover and soybean plates was accompanied by difference in oxidase test results. Bacteria collected from clover tested as oxidase negative, whereas soybean samples were oxidase positive. In these ways our results were different from the expected results. Perhaps we isolated Gram-negative Azotobacter from the clover rather than Rhizobium. Other than these two results, all of the other results were obtained as expected.

The very first Gram stains proved that we had been successful with both the soybean and the clover samples. Gram-negative rods were found in pairs, groups and clusters. Positive test for motility proved that the bacteria have flagella (peritrichous flagella). The negative result for capsule presence gives the idea that maybe the extracellular slime is not really slime layer but closely associated polysaccharide slime that might have sloft off during staining process (thus also explaining the dark clouds seen around the bacteria: see Capsule Stain).

Positive result for catalse test provided further evidence that the bacteria are aerobic with the catalase enzyme present. Both of the samples produced acid without any gas production in the dextrose tube changing the color from red to yellow. The anaerobic chamber test did not work quite as expected. We did not think that any plate would have growth in them when put in anaerobic conditions, but all of the plates had colonies forming in them. This result suggests that maybe the anaerobic chamber was not sealed tightly and therefore these results can be completely discarded.

Ecological Role:
Rhizobium is one of the few nitrogen-fixing bacterium that lives in close symbiotic association with legumes such as peas, beans, alfalfa and clovers. The plant provides carbohydrate to the bacteria and, in return, the bacteria supplies fixed nitrogen compounds to the plant. Rhizobia have an enzyme called nitrogenase that catalyzes the conversion of nitrogen gas to ammonia.

N₂ + 6e^- + 6H⁺ -> 2NH₃

Interesting Characterisitcs:

Nitrogenase becomes inactive in the presence of a large amount of oxygen. Thus Rhizobium lives in nodules so that a microaerobic environment is created. Amount of oxygen in the nodule is controlled through a protein called legaemoglobin (like hemoglobin).
The bacterium produces polysaccharide slime that is not true capsule. It seems like it just swept off of the bacterial surface giving the organism the slimy look.

Significance:
This isolation of Rhizobium allows us to study the organism and understand exactly how it fixes nitrogen. This understanding can help in the field of agriculture. Fertilizers can be made using these bacteria in some way. It helps us understand symbiotic relationships between plants and bacteria. It is important to have a step by step protocol to isolate this bacterium so that bacterium can easily be obtained from the nodules. Small clover root nodules can be easily crushed so it is important to follow a set protocol so that the nodules are not destroyed. A systematic procedure is needed to make sure that the desired organism, Rhizobium, is isolated so that it can be grown on YMA plates.