Lab7 - Nitrogen Symbiotic Fixing by Rhizobium PDF

Ecological and Environmental Microbiology Dr. Abeer Aloufi Ecological and Environmental Microbiology Env 231 Prepared By: Dr. Abeer Salem Aloufi...

Ecological and Environmental Microbiology Dr. Abeer Aloufi Ecological and Environmental Microbiology Env 231 Prepared By: Dr. Abeer Salem Aloufi 0 Ecological and Environmental Microbiology Dr. Abeer Aloufi Seventh lab A- Nitrogen symbiotic Fixing by Rhizobium Nitrogen in the air Nitrogen is required by all living organisms for the synthesis of proteins, nucleic acids, and other nitrogen-containing compounds. The earth’s atmosphere contains almost 80% nitrogen gas. It cannot be used in this form by most living organisms until it has been fixed, that is reduced (combined with hydrogen), to ammonia. Green plants, the main producers of organic matter, use this supply of fixed nitrogen to make proteins that enter and pass through the food chain. Microorganisms (the decomposers) break down the proteins in excretions and dead organisms, releasing ammonium ions. These two processes form part of the nitrogen cycle. The nitrogen cycle The nitrogen cycle is a series of processes that convert nitrogen gas to organic substances and back to nitrogen in nature. It is a continuous cycle that is maintained by the decomposers and nitrogen bacteria. The nitrogen cycle can be broken down into four types of reaction and microorganisms play roles in all of these as the table below shows. 1  Ecological and Environmental Microbiology Dr. Abeer Aloufi Reaction Microorganism Condition Process Nitrogen fixation Nitrogen-fixing aerobic/anaerobic The first step in the synthesis of virtually all nitrogenous bacteria compounds. e.g. Rhizobium Nitrogen gas is fixed into forms other organisms can use. Ammonification Ammonifying aerobic/anaerobic The decomposers, certain soil bacteria and fungi, break (decay) bacteria down proteins in dead organisms and animal wastes (decomposers) releasing ammonium ions which can be converted to other nitrogen compounds. Nitrification Nitrifying Aerobic Nitrification is a two-step process. Ammonia or bacteria ammonium ions are oxidized first to nitrites and then to e.g. nitrates, which is the form most usable by plants. Nitrosomonas & Nitrobacter Denitrification Denitrifying Anaerobic Nitrates are reduced to nitrogen gas, returning nitrogen to the air and completing the cycle. bacteria Nitrogen fixation Nitrogen can be fixed in three ways: 1. Atmospheric fixation: this occurs spontaneously due to lightning; a small amount only is fixed this way. 2. Industrial fixation: the Haber process, which is very energy inefficient, is used to make nitrogen fertilizers. 3. Biological fixation: nitrogen-fixing bacteria fix 60% of nitrogen gas. Biological fixation: The reduction of nitrogen gas to ammonia is energy intensive. It requires 16 molecules of ATP and a complex set of enzymes to break the nitrogen bonds so that it can combine with hydrogen. Its reduction can be written as: energy N2+3H2 2NH3 Fixed nitrogen is made available to plants by the death and lysis of free- living nitrogen-fixing bacteria or from the symbiotic association of some nitrogen-fixing bacteria with plants. Examples of nitrogen-fixing bacteria are shown in the table below: 2 Ecological and Environmental Microbiology Dr. Abeer Aloufi Nitrogen-fixing bacteria Free-living Symbiotic association with plants Aerobic Anaerobic Legumes Non-legumes e.g., peas, beans e.g., alder tree Azotobacter Clostridium Rhizobium Frankia Rhizobium Rhizobium is the most well-known species of a group of bacteria that acts as the primary symbiotic fixer of nitrogen. These bacteria can infect the roots of leguminous plants, leading to the formation of lumps or nodules where the nitrogen fixation takes place. The bacterium’s enzyme system supplies a constant source of reduced nitrogen to the host plant and the plant furnishes nutrients and energy for the activities of the bacterium. About 90% of legumes can become nodulated. In the soil the bacteria are free-living and motile, feeding on the remains of dead organisms. Free-living Rhizobia cannot fix nitrogen and they have a different shape from the bacteria found in root nodules. They are regular in structure, appearing as straight rods; in root nodules, the nitrogen-fixing form exists as irregular cells called Bacteroides which are often club and Y-shaped. Rhizobium is bacilli bacteria, non-spore-forming, motile, and aerobic. 1- Rhizobium is free living in the soil. 2- Penetration of Rhizobia into deformed root hair. 3- Secretion of different things e.g., Flavonoids. 4- The bacteria show different shapes like T-Y-L-X-V, this phase is called Bacteroid. 3 Ecological and Environmental Microbiology Dr. Abeer Aloufi Name of experiment: Nitrogen symbiotic Fixing by Rhizobium. Aim of experiment: Determine and identify the nitrogen symbiotic fixing bacteria by using a microscope. Material: Root nodule containing bacteria. sterile distilled water. Petri dish. Sterile tongs. Flam. Dettol. Cotton. Method: 1- Open the flam, with cotton and Dettol clean up the work area (the bench). 2- Separate the root nodule from the root. 3- Wash the nodule with water-containing drops of antiseptic to avoid contamination. 4- Put the nodule in a flask containing 95% of Ethanol for 30-60 seconds. 5- Move the nodule by sterile tongs to a tube containing sterile distilled water. 4 Ecological and Environmental Microbiology Dr. Abeer Aloufi 6- Mash the nodule in a Petri dish then prepare a bacterial smear after that stain the bacteria by using Gram stain and examine of bacteria under the microscope. Result and conclusion: 5