EXERCISE 6 dinsicctaniny SOIL MICROBIOLOGY “Experiment 6.1 Enumeration of Soil Microorganisms Experiment 6.2. Nitrogen Cycle BACKGROUND Soils are complex environments in which microorganisms play a major role. For example, the soil microbial community is a source of nutrients, contributing to nutrient cycling and decomposition. Complex microbial interactions with other microorganisms, macroorganisms and nutrients influence degradation processes. These organisms interact with each other and with plants in the formation and maintenance of soils Unfortunately, most microorganisms in soils have not been cultured and studied. Nevertheless, life on earth could not be sustained in the absence of the soil microbiota and their many important interactions. Actinomycetes (including actinoplanetes, nocardioforms, and streptomycetes), other bacteria and filamentous fungi (Rhizopus, Mucor, Penicillium and Aspergillus) are all important members of the soil microbial community. Other organisms include protozoa, algae, cyanobacteria, nematodes, insects, and other invertebrates and viruses but these will not be examined in this exercise. Each gram of rich garden soil may contain millions of these micro- and macroorgarisms. The types and numbers of microorganisms found in the soil depend on the environmental conditions in the soil. By their growth and activities, these microorganisms modify the soil. One of the most important modifications is mineralization of soil organic material to release its carbon, nitrogen, phosphorus and ‘sulfur atoms. Mineralization makes these atoms available to the microorganism. For example, nitrogen from proteins and other compounds can be converted into a number of inorganic compounds. During mineralization, the nitrogen present in the proteins is. first released in the form of amino acids by extracellular proteases. The amino acids are then degraded by enzymes, which carry out decarboxylation, deamination or removal of side chains. The nitrogen is released as ammonia, which can be used by plants and microorganisms for protein synthesis. Ammonia can also be converted by microorganisms to nitrite, nitrate and nitrogen gas (N2). All of these chemical reactions are collectively referred to as the nitrogen cycle. The nitrogen cycle is shown in Figure 61 26eB Figure 6.1 The Nitrogen Cycle. Flows that occur predominantly under aerobic conditions are noted with open arrows arrows. Anaerobic processes are indicated with solid thick arrows. Other processes occur under both aerobic and anaerobic conditions. Important genera contributing to the nitrogen cycle are given as examples, Aesimilatoy ivitoaton (ntrobactr, Imtrococcus) (any gener) Penngaton sich AAI 1, «1:0 No; ton (etobacter Costa, Geobacter metteducens ' petopnhe bacter) Desutonon na ostium tiation iorkzaton ee ‘trosococcus) ” LEARNING OBJECTIVES: 1. Be able to quantify soil microorganisms. 2. Be able to demonstrate the role of soil microorganisms in the nitrogen cycle 3. Demonstrate the liberation of ammonia from nitrogenous organic compounds by microorganisms 4. Demonstrate the enzymatic conversion of ammonia to nitrites by microorganisms 5. Demonstrate the reduction of nitrates to nitrogen gas by microorganisms 6. Differentiate between symbiotic and nonsymbiatic nitrogen fixation SUGGESTED READING IN THE TEXTBOOK: Chapters 25. 27Experiment 6.1. Enumeration of Soil Microorganisms Since soils vary greatly with respect to their physical features (e.g. pH, type, temperature and other related factors), the microorganisms present will also vary. For example, acid soils will have a higher number of fungi compared to alkaline soils and rich garden soil will contain more actinomycetes than either the other bacteria or fungi. Not surprisingly, no single technique is available to count the microbial diversity found in average garden soil. ao ® cD In this experiment, the relative number of fungi,actinomycetes and other bacteria in a sample of garden soil will be determinetusing the serial dilution agar Plating procedure and selective media. To support the three different groups of microorganisms three different media will be used. Sabourand dextrose agar will be used for the isolation of fungi. Glycerol yeast agar-will be used for the isolation of inom: ind Wyplic Soy agar Tor the isolation of other bacteria. Material Procedure: 1. Place 1 grgm of soil into 99 mL of sterile water. Mix thoroughly for 3 minutes. ‘This is your 107 dilution. Prepare a 10% and 10° dilution using the other two 99 mL bottles of sterile water. 2. Label three sets of four empty plates as follows: actinomycetes (10%, 10%, 105, 10°), fungi (10%, 10°, 10%, 10°) and other bacteria (10%, 10°, 10°, 10”). 3. Using a 1 mL pipette and aseptic technique distribute the proper volume of each soil dilution to the respective petri dish as outlined in Figure 7.2 4. Now pour approximately 15-20 mL of glycerol yeast agar into the actinomycetes plates, swirl on a flat surface and allow to harden. Repeat procedure with Sabourand agar for the fungi and the tryptic soy agar for the other bacteria. 5. Invert plates and incubate for 3 - 7 days at room temperature. 28Figure 6.2 Scheme for using the Plate Count Procedure for Enumerating Soil Microorganisms. Aetinomyostes i Next Week? 6. Observe all the plates and note the appearance of the colonies. Count the dilution with between 30 and 300 and calculate the number of respective organisms per gram of soil. 29Experiment 6.2 Nitrogen Cycle om part A: Ammonification part B: Nitrification part C: Denitrification part D: Nitrogen Fixation One of the phases of the nitrogen cycle is ammonification - the release of ammonia (NHs) from organic compounds by deamination. The proteases and other enzymes that accomplish ammonification are produced by soil bacteria (e.g., Bacillus, Clostridium, Proteus, Pseudomonas, Streptomyces). Once ammoria is released into the soil, it dissolves in water to form ammonium ions as follows: NHs + HO - NHOH — NH. + OH Ammonia Water Ammonium Ammonium Hydroxide hydroxide ion ion Some of the ammonium ions are then used by plants and microorganisms for the synthesis of amino acids. Peptone broth can be used as an organic nitrogen substrate in order to examine the ability of different bacteria and microorganisms in soil samples to degrade the organic nitrogen and release ammonia. Ammonia can be detected by adding Nessler’s reagent to a broth culture containing the respective microorganisms. If ammonia is present, the culture will turn yellow to yellqwis wn. The deeper the coloration, the more ammonia is produced. NS anita oe © BaciwS cereus 7 @ proteus, VulognS @ Ve pseudomons Aluprscens -5 empty West fubes Fit patios G)_one SOi\ somple Bacterial strains: @ Last as « contyol. —-Bacillus cereus (ATCC 21768 or 14893) | ) —~Proteus vulgaris (ATCC 13315) “\) — Pseudomonas fluorescens (ATCC 13525) > ) Procedure: 1. Label Shelve peptone tube9, with the names of the bacteria strains, one with the ‘soil samp! iS last BS a control. x ene : — 30B 2. Inoculate each tube with several loopfuls of the appropriate culture. For the soil isang, sample pick up several loopfuls of soil. Do not inoculate the control. 3. Incubate the tubes for 7 days at room temperature. Next Week: 4, Remove 1 ml from each tube and place in an empty test tube. Add several drops of Nessler's reagent. Mix. If the culture does not change color no ammonia is present; if a pale yellow color appears, a small amount of ammonia was produced; if the color is deep yellow, a moderate amount of ammonia was'produced; and if a brown color or precipitate appears, a large amount of ammonia was produced. 5. Which bacteria made the most ammonia? the least?) Race mie Osea PART B: Nitrification In an.getobie environment, arnmonia is liberated into the soil by the mmonification part of the nitrogen cycle but does not accumulate. Instead, if it is not rectly used as a nitrogen source by either plants or microorganisms, it is oxidized to nitrates by a two-step process called nitrification. Nitrification primarily involves two genera of chemolithoautotrophic soil bacteria: Nitrosomonas and Nitrobacter. The chemical tranformations of nitrification are as follows: Nitrite formation (step 1) Nitrosomonas NH,” + 11/202 > NOS + 2H" + H20. Ammonium oxygen Nitrite Hydrogen Water ion ion ion Nitrate formation (step 2) Nitrobacter NO; + 1/202 > NOs Nitrite Oxygen Nitrate ion The nitrate released into the soil is highly soluble and can be easily be assimilated by plants and Some microorganisms for the synthesis of proteins. Unfortunately, it is also readily leached form the soil, thus lowering soil fertility. 7 31~ Ammonium sulfate broth can be used to demonstrate the ability of garden soil microorganisms to oxidize ammonia to nitrite and nitrite broth to demonstrate the further oxidation of nitrite to nitrate. Materials: nontwk7 -garden soil, al ammonium sulfate bottles (20mL) cnittité broth bottles (20mL) Bh ccczmae . =Nessler's reagent Spot plate ~Trorhmsdorfs reagent -diphenylamine -sulfuric agid (1 part concentrated sulfuric acid to 3 parts water} “A ml pip F 2 Procedure: ay 1. Inoculate the ammonium sulfate broth bottle with a loopful of soil. Do the same eG * the nitrite broth bottle. Shake the bottles vigorously for 3 minutes. 2. Incubate the bottles the bottles at room temperature for 7 days. i oo we wh eS 3. Using a spot'plate, place a drop of sulfuric acid-and 3 drops of —- reagent if.a well. Add a Sop of ulus med from the ammoniui using a Pasteur pipette and mix. A blue black color indicates the presence no color change is indicative of the absence of nitrite. —a Next Week Sarto»eee. PART C: Denitrification Denitrification is the reduction of nitrates to nitrites and eventually to nitrogen gas, as follows: NOs -—+ NOZ -> N20 —> Ne Nitrate Nitrite Nitrous Nitrogen ion ion oxide gas The bacteria (e.g. Bacillus, Alcaligenes, Pseudomonas) that accomplish the above are referred to as denitrifying bacteria. Once denitrification has occurred, nitrogen is removed from the nitrogen cycle since nitrous oxide and nitrogen gas are not usable by most organisms. Because O: is not necessary for denitrification to occur, this form of anaerobic respiration in which the nitrates serve as electron acceptors for the denitrifying bacteria in their energy metabolism. Denitrification occurs most rapidly in waterlogged anaerobic soils Medium containing a nitrate substrate for gas formation and a Durham tube for the detection of the gas. If gas formation occurs, a bubble will appear in the Durham tube; if no bubble appears, gas formation (denitrification) has not occurred. Materials: v n soil ate broth tubes containing Durham tubes -2 nitfate“free broth tubes containing Durham tubes -o-naphthylamine reagent -sulfanilic acid -powdered Zinc Wm pipettes © Bacteri; ae “Pseudomonas Muorescend (ATCC 13525) broth culture Procedure: 1. Inoculate one tube of nitrate broth containing the Durham tube with a loopful of soil. Inoculate the other tube with a loopful of Ps. fluorescens 2. Repeat step 1 with the nitrate-free broth containing the Durham tubes. 3. Incubate all the tubes at room temperature for 7 days. Do not shake during incubation. 33Next Week: 4. Observe the tubes for gas formation. 5. To test for nitrites, add 1 mL of a-naphthylamine reagent and 1 mL of sulfanilic acid to each tube and mix. The development of a red color within 30 seconds indicates that nitrites are present. 6. If any of the tubes fail to develop a red color that could mean that it still has its full original nitrate supply or it could have undergone denitrification, with nitrite being further converted to nitrogen. To distinguish between these possibilities, a pinch of powdered zinc can be added to any tube that failed to turn red. The zinc catalyzes the reduction of nitrate to nitrite and produces a red color within minutes if nitrate is present. Lack of a red color indicates the absence of nitrate. Jen gfe to ammonia. Miggoorganis canfffx nitrogen under anaerobic offiattons No : Nitrogen ‘ gas : pe nitrogen-fixing, non: foorganisms\(¢. .g. Azptobacter, Clostridium, and ith the ror of gi vatiot th feria} es in the rhizosphere (the g., Rhizobium); however, , arse a more important fole a nitfoign fixation pe symbiotic associations withthe roots of eguimes (gala lupip soyppans, weet peas). When Rhizdbium infects a toot 3 fant. This root nodule spifixation tc occur. The jst plant. During their lable to the plant. provides the anaerot symbiotic Rhizobi growth, they fix ga {Azotobacter jzobium can be found in the root nig of avail lation of Azotobacter is possible becap f nitrogen-fixing bacteria. f | yf / 34