8. ANALYSIS OF THE MICROBIOLOGICAL QUALITY OF DIFFERENT ENVIRONMENTS
8.3. Examination of microorganisms participating in the nitrogen cycle
Nitrogen occurs in nature in different oxidation states, mostly in the molecules transformed by different living or-ganisms (Fig. 45). Many of the processes are catalysed only by microoror-ganisms.
Fig. 45. The nitrogen cycle.Nitrogen compounds areshown in rectangles. The different nitrogen transformation processes are indicated with arrows.
Biological nitrogen fixation is carried out by prokaryotic organisms only. During the process of nitrogen fixation, the oxidation state of the nitrogen atom is reduced from 0 (nitrogen gas) to -3 (ammonia). The ammonia gained by nitrogen fixation can later be assimilated by organic compounds.
Nitrogen-fixing organisms can be divided into two major groups: free-living organisms (e.g. Azotobacter, Clostridium,Anabaena) and symbionts of higher/other organisms (close association). The latter group consists
mostly of the members of the genusRhizobium, which are associated with leguminous plants (e.g. peas, beans, soybean). Free-living rods infect the root hairs of leguminous plants. The plant responds by producing nodules to seal off the infection. Inside these swellings,Rhizobiumcells grow and become pleomorphic symbionts called bacteroids. Bacteroids have characteristic morphology and they fix atmospheric nitrogen.
Among nitrogen-fixing prokaryotes, aerobic, microaerophilic and anaerobic microorganisms can be identified.
Since the nitrogenase enzyme is very sensitive to the presence of oxygen, the intracellular oxygen partial pressure should be reduced in an aerobic/oxic environment. The nodules contain leg-hemoglobin, which is responsible for the suitably low oxygen tension necessary for bacteroids (Fig. 46).
Fig. 46. Morphology of bacteroids.(a) Bacteroid-containing plant cell inside the nodule. (b) Bacteroids stained with methylene-blue.
EXERCISE 90: EXAMINATION OF BACTEROID MORPHOLOGY Object of study, test organisms:
bacteroids in the nodule of leguminous plants Materials and equipment:
root sample (leguminous plant) scalpel
forceps glass slide
methylene-blue solution Bunsen burner
light microscope Practise:
1. Cut a nodule off the washed leguminous root sample using forceps and a scalpel (The active nodules have a slightly pink colour, since they contain leg-hemoglobin).
2. Gently press the nodules between two glass slides.
3. Air dry and heat fix the smear, and then stain the smear with methylene blue (see EXERCISE 36).
4. Observe the large, irregular rods of the bacteroids under the microscope and draw their morphology.
EXERCISE 91: STUDY OF CYANOBACTERIA WITH HETEROCYSTS OCCURRING IN NATURAL WATER SAMPLES
In some of the free living cyanobacteria (Fig. 47) (e.g.Cylindrospermopsis raciborskii, Nostoc punctiforme), ni-trogen fixation takes place in thick-walled, specialised nini-trogen-fixing cells called heterocysts.
Fig. 47. Filamentous nitrogen-fixing cyanobacteria from lake water samples.(a)Anabaenasp. (b) Aphanizo-menonsp. (c)Cylindrospermopsis raciborskii(Micrographs by Lajos Vörös and Boglárka Somogyi). Arrows
in-dicate the heterocysts, while star inin-dicates akinete.
Object of study, test organisms:
Free-living cyanobacteria Materials and equipment:
water sample (e.g. lake, aquarium, etc.) Pasteur pipette
glass slide cover slip microscope Practise:
1. Put a drop of water sample onto a slide and cover with a cover slip.
2. Check and draw the morphology of different cyanobacteria (unicellular, filamentous, etc.) and try to search for heterocysts in the microscopic field.
EXERCISE 92: DEMONSTRATION OF AMMONIFICATION
During ammonification, some organic nitrogen compounds (amino acids, carbamide, etc.) are deaminated to form ammonium ions and as a function of pH, ammonia is released into the environment. For such deamination reactions, bacteria use many enzymes with different substrate specificity (one way of getting rid of excess organic nitrogen).
The remaining part of the molecule can be used for e.g. energy generation. Within the nitrogen cycle, ammonific-ation is considered as a mineralisammonific-ation step. Ammonia and ammonium ion can be taken up and used for amino acid synthesis by other organisms (plants, microbes) or it can be absorbed in the soil by humus-colloids (Fig. 45).
Object of study, test organisms:
unknown bacterial slant culture Materials and equipment:
peptone broth (see Appendix) urea broth (see Appendix) inoculating loop
sterile distilled water in test tubes pipette, sterile pipette tips Bunsen burner
incubator
Nessler’s reagent (see Appendix) Practise:
1. Make a suspension from the unknown bacterial strain in sterile distilled water.
2. Inoculate the peptone/urea broth tubes with 0.1-0.1 mL bacterial suspension.
3. Incubate the tubes at 28°C for one week.
4. The presence of accumulated ammonia in the broth can be demonstrated by adding a few drops of Nessler’s reagent (an alkaline solution of potassium-tetraiodo-mrcurate). Weak positive reaction yields a yellow colour, strong positive reaction results in a yellowish brown colour and precipitation (basic mrcury-amido-iodide).
EXERCISE 93: DEMONSTRATION OF NITRIFICATION AND INHIBITION OF NITRIFICATION
Under aerobic conditions, ammonia does not accumulate in the soil or water. Beside the assimilation of ammonia (for e.g. amino acid synthesis), certain bacteria can gain energy by utilizing ammonia as electron donor (as well as generating reducing potential) in dissimilative processes. Nitrification is carried out by chemolithoautotrophic bacteria (e.g.NitrosomonasandNitrobacter) and different heterotrophic microorganisms. Nitrate can be taken up by plants more easily than ammonia, however, due to its higher degree of mobility, nitrate can be leached out from soils, deteriorating the quality of both surface and underground waters.
Nitrification is a two-step biological process(Table 9).
Table 9. Steps of nitrification
Typical bacterial genus
The key enzyme of ammonia-oxidation is ammonia monooxigenase (AMO), which is usually active in the presence of copper ions. Allyl-thiourea (ATU) is a selective copper-chelator compound, thus ATU can inhibit the ammonia monooxigenase enzyme.
Object of study, test organisms:
nitrifying bacteria in soil Materials and equipment:
ammonia broth (see Appendix) nitrite broth (see Appendix) soil sample
sterile distilled water in test tubes pipette, sterile pipette tips
Nessler’s reagent (see Appendix) Zn powder
Practise:
1. Add ATU at a final concentration of 5 mg/L to half of the nutrient tubes containing ammonia and nitrite broth.
2. Inoculate the media with 0.5 mL soil suspension.
3. Incubate the samples at 28°C for one week.
4. Transfer 1-1 mL of each broth to a new, empty test tube. Add some drops of Griess-Ilosvay-reagent to the tubes (nitrite A and B reagent). Mix the contents of the tubes. The presence of nitrite in the broth is demonstrated by a cherry red colour within 30 sec.
5. There are two possibilities if there is no red coloration: either ammonia oxidation has not taken place (effect of the inhibitor or the absence of ammonia-oxidizing bacteria), or nitrite was completely oxidised to nitrate. To distinguish between these two options, add a small amount of zinc powder to the test tubes (max. 5 mg/mL). If nitrate has been formed during nitrification, zinc will reduce it back to nitrite, and the Griess-Ilosvay-reagent will turn red.
6. If there is still no colour change after the addition of zinc to the ammonium broth, add Nessler’s reagent to the remaining broth. If a yellowish brown colour develops, it indicates that ammonium ions remain in the broth.
7. Summarise your results in a table form indicating the compound detected and the type of oxidation in each media (see Appendix).
EXERCISE 94: DEMONSTRATION OF DISSIMILATORY NITRATE REDUCTION
During the dissimilatory nitrate reduction, the end products are nitrite, dinitrogen-oxide and nitrogen gas. The process is anaerobic and takes place in compacted environments, such as water-saturated soils, river or lake sedi-ments, inside the gastrointestinal tract of higher organisms. When a gaseous substance (N2O, NO, N2) is produced, the process is called denitrification. This process plays a very important role in the nitrogen equilibrium and self-purification of soil and water. From a biochemical point of view, this process is nitrate reduction: the utilisation of nitrate as electron acceptor for the biological oxidation of different organic and inorganic (e.g. H2S) substances.
Object of study, test organisms:
nitrate-reducing and denitrifying bacteria of soil unknown bacterial strain slant culture
Materials and equipment:
soil/sediment sample
nitrate broth containing Durham tubes (see Appendix) sterile distilled water in test tubes
pipette, sterile pipette tips vortex mixer
incubator empty test tubes
Griess-Ilosvay reagent (see Appendix) Nessler’s reagent (see Appendix) Zn powder
Practise:
1. Prepare suspension from the soil/sediment sample as well as from bacterial cultures in sterile distilled water.
2. Inoculate the nitrate media with 0.5 mL soil/sediment/bacterium suspension.
3. Incubate the samples at 28°C for one week.
4. Evaluate the results for the presence of the following products (see also EXERCISE 93):
Griess-Ilosvay reagent Nitrite (NO2-):
Bubbles inside the Durham tubes Nitrogen gas (N2):
Nessler’s reagent Ammonia: