General purposes:1- To make the students aware with the role of microbes in maintaining environment, existing microbial interactions and recycling of nutrients in nature:2- A technique for the isolation of a free living soil bacterium Azotobacter.3- A technique for the isolation of root nodule Bacterium Rhizobium sp.
By traveling through one of the four processes in the Nitrogen Cycle! (1) Nitrogen Fixation (4) Denitrification Nitrogen Cycle(3) Nitrification (2) Ammonification (mineralization)
Nutrients- The Nitrogen Cycle•modified from Goldman and Horne. 1994. Limnology. McGraw Hill.
N N N Why does N atmosphericnitrogen need to be converted? N N
It is one of nature’s great ironies…Nitrogen is an essential component of DNA, RNA,and proteins—the building blocks of life.why is N fixation important?• atmospheric N2 is inert – biotically unavailable.• availability of fixed N is often the factor mostlimiting to plant growth
How could atmospheric nitrogenbe changed into a form that canbe used by most living organisms? N N
There are three ways that nitrogen could be “fixed”!(a) Atmospheric Fixation(b) Industrial Fixation(c) Biological Fixation Bacteria
Atmospheric Fixation Lightning “fixes” Nitrogen!(Only 5 to 8% of the FixationProcess)The enormous energy of N N Olightning breaks nitrogen Nitrogen combinesmolecules apart and enables with Oxygenthe nitrogen atoms to combinewith oxygen forming nitrogen Nitrogen oxides formsoxides (N2O). Nitrogen oxides (N2O) Nitrogendissolve in rain, forming (NO3) oxides dissolvenitrates. Nitrates (NO3) are in rain and change tocarried to the ground with the nitratesrain. Plants use nitrates to grow!
Industrial Fixation NN H Under great pressure, at atemperature of 600 degrees NCelsius, and with the use of H3 a catalyst, atmospheric nitrogen (N2) and hydrogen Industrial Plant combines are combined to form nitrogen and hydrogen ammonia (NH3). Ammonia can be used as a fertilizer. (NH3) Ammonia is formed Ammonia is used a fertilizer in soil
3. Biological Fixation:a. Non-symbiotic bacteria) Free Living Bacteria: (“fixes” 30% of N2) Highly specialized bacteria live in the soil and have the ability to combine atmospheric nitrogen with hydrogen to make ammonia (NH3).Such as Azotobacteraceaeb.Symbiotic Relationship Bacteria: (“fixes” 70% of N2)Bacteria live in the roots of legume family plants and provide the plants with ammonia (NH3).Among the most beneficial microorganisms of the soil are those that are able to convert gaseous nitrogen of the air to “fixed forms” of nitrogen that can be utilized by other bacteria and plants. Without these nitrogen-fixers, life on this planet is probably disappear within a relatively short period of time. The utilization of free nitrogen gas by fixation can be accomplished by organisms that are able to produce the essential enzyme nitrogenase. This enzyme, in the presence of traces of molybdenum, enables the organisms to combine atmospheric nitrogen with other elements to form organic compounds in living cells.Such as Rhizobiaceae.Other organisms of less importance that have this ability are a few strains of Klebsiella, some species of Clostridium, the cyanobacteria, and photosynthetic bacteria. In this exercise we will concern ourselves with two activities: the isolation of Azotobacter from garden soil and the demonstration of Rhizobium in root nodules of legumes.
Biological Fixation There are two types of “Nitrogen Fixing Bacteria”Free Living Bacteria Symbiotic Relationship Bacteria(“fixes” 30% of N2) (“fixes” 70% of N2)
Free Living Bacteria Highly specialized bacteria live in the soil and have theability to combine atmospheric nitrogen with hydrogen to make ammonia (NH3). N N H NH3 Free-living bacteria live in soil and combine atmospheric nitrogen with hydrogen (NH3) Nitrogen changes into ammonia Bacteria
Symbiotic Relationship Legume plants BacteriaBacteria live in the roots oflegume family plants andprovide the plants with Nammonia (NH3) in exchangefor the plant’s carbon and a protected-home. NH3 N Roots with nodules where bacteria live Nitrogen changes into ammonia.
Nitrogen FixationThe nodules on the rootsof this bean containbacteria calledRhizobium that helps byconverting nitrogen inthe soil into a form theplant can utilize it. 14
Mechanism of N-fixation:The general chemical reaction for the fixation of nitrogen (N + 3H2 + Energy -> 2NH3) is identical for both the chemical and the biological processes. The triple bond of N must be broken and three atoms of hydrogen must be added to each of the nitrogen atoms. Living organisms use energy derived from the oxidation ("burning") of carbohydrates to reduce molecular nitrogen (N2) to ammonia (NH3).• AZOTOBACTERACEAEAzotobacteraceae that fix nitrogen as free-living organisms under aerobic conditions: Azotobacter and Azomonas. Both are large gram-negative motile rods that may be ovoid or coccoidal in shape, (pleomorphic). The free-living Azotobacteraceae are beneficial nitrogen-fixers, their contribution to nitrogen enrichment of the soil is limited due to the fact that they would rather utilize NH3 in soil than fix nitrogen. In other words, if ammonia is present in the soil, nitrogen fixation by these organisms is suppressed.• RHIZOBIACEAEThe symbiotic nitrogen-fixers of genus Rhizobium, family Rhizobiaceae, are the principal nitrogen enrichers of soil. Three genera in family Rhizobiaceae: Rhizobium, Bradyrhizobium, and Agrobacterium. Although the three genera are related, only genus Rhizobium fixes nitrogen. This genus of symbiotic nitrogen-fixers contains only three species:• R. leguminosarum: peas, beans.• R. meliloti: sweet clover.• R. loti: trefoil.• All three of these species are gram-negative pleomorphic rods (bacteroids), often X-, Y-, star-, and clubshaped; some exhibit branching. All are aerobic and motile.
Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium) Free living Symbiotic with plants Anaerobic (Winogradsky Aerobic Legumes Other plants column) Clostridium (some) Azotobacter Desulfovibrio Beijerinckia Frankia Purple sulphur bacteria* Rhizobium Klebsiella (some) Azospirillum Purple non-sulphur bacteria*Cyanobacteria (some)* Green sulphur bacteria*
Procedure for isolation of AZOTOBACTERACEAEFIRST PERIOD (ENRICHMENT)Proceed as follows to inoculate a bottle of the nitrogen free glucose medium with a sample of garden soil.Materials:• 1 bottle (50 ml) N2-free glucose medium (Thompson-Skerman) or Azotobacter agar• rich garden soil (neutral or alkaline)• spatula1. with a small spatula put about 1 gm of soil into the bottle of medium. Cap the bottle and shake it sufficiently to mix the soil and medium.2. Loosen the cap slightly and incubate the bottle at 30° C for 4 to 7 days. Since the organisms are strict aerobes, it is best to incubate the bottle horizontally to provide maximum surface exposure to air.SECOND PERIOD (PLATING OUT)During this period a slide will be made to make certain that organisms have grown on the medium. If the culture has been successful, a streak plate will be made on nitrogen-free, iron-free agar. Proceed as follows:Materials:Microscope slides and cover glasses microscope, 1 agar plate of nitrogen-free, iron-free glucose medium1. After 4 to 7 days incubation, carefully move the bottle of medium to your desktop without agitating the culture.2. Make a wet mount slide with a few loopfuls from the surface of the medium and examine under oil immersion, Look for large ovoid to rod-shaped organisms, singly and in pairs.3. If the presence of azotobacter-like organisms is confirmed, streak an agar plate of nitrogen-free, iron-free medium, using a good isolation streak pattern. Ferrous sulfate has been left out of this medium to facilitate the detection of water-soluble pigments.4. Incubate the plate at 30° C for 4 or 5 days.
Azotobactereace on different media : a) Brown-agar medium, b)Winogradsky solution, c) smoothed soil paste–plate surface, d)mannitol-agar, e ,f, g, h) differential LG agar medium (differentspecies and components.
Procedure for isolation for isolation of RHIZOBIACEAE:• Materials:1. washed nodules from the root of a legume2. methylene blue stain3. microscope slides – pink nodules were selected from the root of a legume and washed by water, then kept in (MgCl2) for period of time, and washed again by water – Place a nodule on a clean microscope slide and crush it by pressing another slide over it. Produce a thin smear by sliding the top slide over the lower one. – After air-drying and fixing with heat, stain the smear with methylene blue for 30 seconds. – Examine under oil immersion.
A. Questions:1. What enzyme is responsible for nitrogen fixation? By which mechanism level of O2 regulated to obtain maximum nitoginase activity?2. Why is nitrogen fixation so important?3. from the standpoint of amount of nitrogen fixation, is this group of nitrogen-fixers Rizobacteriaceae more or less important than the Azotobacteraceae?4. On your opinion does it possible to increase fixation in unamended soil by addition of high populations of bacteria (soil inoculation)?5. Draw some of the organisms on the Laboratory Report. Look for typical bacteroids of various configurations.