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2 Course Title: Agricultural Microbiology Course Code: Plsc1014 and HORT1045 Credit hours: 3(2+3); ECTS: 5 for Plant Science department Students and : 3(1+3); ECTS: for Horticulture department students Program: Degree Delivered to: 1 st Year Plant Science and Horticulture Students Academic Year: 2016/17 Semester: Two (II) Instructor: Derib Alemu (M. Sc.) Course description: Definition and historical development of Microbiology, types and structure of microscopes; culture techniques; classification of microorganisms into different groups; characteristics of bacteria, microbes in rhizosphere and phyllosphere; morphology, biology, nutrition, reproduction and classification of bacteria, fungi, viruses and other microorganisms of agricultural importance; Microbiology of plant pathogenic microbes; role of different microbes in nutrient transformations and nitrogen fixation; microbial interaction in the soil system; composting and decomposition of organic matter through microbes; mycorrhizae and their role in agriculture; commercial use of biofertilizers in agriculture; role of microorganisms in soil fertility and crop production cycles; bioremediation of contaminated soils and biopesticides. Course objectives: The objective of the course is to acquaint students with microorganisms of agricultural importance and techniques of their handling At the end of the course students will be able to describe:- Micro-organisms of agricultural importance and techniques of their handling Importance of agricultural microbiology; Microorganisms of agricultural importance: bacteria, fungi and viruses; Laboratory techniques for culturing and identification of microorganisms; Roles of microorganisms in agriculture. 2 P a g e Compiled by: Derib A.

3 3 P a g e Compiled by: Derib A. TABLE OF CONTENTS Contents Page TABLE OF CONTENTS INTRODUCTION Definition and Scope of Microbiology Discovery and Historical Development of Microbiology Theories on the origin of life The germ theory of disease (Koch's Postulates) Culture media and development of pure culture Solidifying agent: Agar BASIC CONCEPTS IN MICROSCOPY Types of Microscopy Preparation and Fixation for Light Microscopic Examination TAXONOMY OF MICROBES Eukaryotic Microbes Prokaryotic Microbes THE STRUCTURE AND FUNCTIONS OF PROKARYOTIC (BACTERIAL) CELLS General Characteristics of Bacteria Bacterial Cell External structure Internal structure MICROBIAL GROWTH AND NUTRITION Microbial Growth Bacterial growth and reproduction The growth curve Measurements of microbial growth (Bacterial counts) Nutrient Requirement Environmental Requirement MICROBIOLOGY OF PLANT PATHOGENIC MICROORGANISMS Causes of Plant Disease Fungi Bacteria Viruses SOIL MICROORGANISMS (Group Assignments) MICROBIAL INTERACTION IN SOIL SYSTEM Plant-Microbe Interaction Aboveground microbe-plant interaction (Phyllosphere or Phylloplane) Below ground microbe-plant interaction (Rhizosphere or Rhizoplane) ROLE OF MICROORGANISMS IN SOIL FERTILITY AND CROP PRODUCTION CYCLES Microbial Metabolism Carbon Transformation and Soil Organic Matter Formation Organic Matter Decomposition and the Soil Food Web Soil organic matter Decomposition process Beneficial Property of Soil Organic Matter and Humus to Soil Nitrogen Cycle Phosphorus Cycle Sulfur Cycle BIOREMEDIATION OF CONTAMINATED SOIL Approaches to Bioremediation Advantages of Bioremediation Disadvantages of Bioremediation BIOPESTICIDES...58

4 Practical/lab Orientation of students about lab safety issues and introducing the common tools (Microscopy etc) used in Microbiology laboratory Preparation of culture/nutrient media for microorganisms Isolation of microorganisms from soil and enumeration of them using the serial dilution plate technique Smear preparation and gram staining of selected bacterial species Observation of certain fungal structures under the microscope (Fusarium, Rhizopus, Pencil lium, Aspergillus etc) Observation of mush room production in the laboratory Activities/Tasks of students Listen to lectures and take short notes. Participating in asking and answering questions Take part in reading assignments Discussing during lab/practical class and writing report Mode of Delivery: The mode of the delivery of the course combines lectures, laboratory/practical activities, discussion, questioning and answering, readings, assignments, individual and/or group works and presentation. Assessment Methods: Evaluation will be carried based on continuous assessment which comprises relevant tests, assignments, seminar and laboratory works and final examination. SUMMARY OF COURSE ASSIGNMENTS Evaluation Point (%) Tentative Time Schedule Individual Assignment and Lab th -12 th Weeks reports Group Assignment 10 4 th -6 th Weeks Quizzes 10 When Necessary Tests th Weeks Class Activity and Attendance 5 When Necessary Final Exam th Weeks Total P a g e Compiled by: Derib A.

5 Course Policy Academic dishonesty, Cheating and plagiarisms: All students are expected to abide by the code of conduct of articles the Senate Legislation of the University throughout this course. Academic dishonesty, including cheating, and plagiarism will not be tolerated and will be reported to concerned bodies for action. If you need to read it, you can get a copy of it from your department (to be copied by yourself). Course approach by student: Class activities will vary day to day, ranging from lectures to discussions. Students should be active participants in this course. You need to ask questions and raise important issues. You should do all the assignments you are supposed to get done. You are required to submit and present the assignment provided within the specified time. Reading assignments will appear on major examinations. So please perform as per the given assignment conditionality s. All issues discussed in class or derived from other sources (provided to you to read) may be the subject of assignment or final exam question items. Please follow the instructions to complete all the assignments provided whether they are to be performed individually or in group. You are responsible for all class announcements and changes. Note on class attendance and participation: You are expected to attend class and lab regularly. I will take attendance on random days during the semester to ensure that students are coming to class/lab, and if you miss class repeatedly, your grade will be affected negatively. If you miss more than 15% of the class attendance you will not sit for final exam. I will often ask questions during my lectures and active participation in class is essential. Cell phones: Cell phones must be silent before entering the class as they are disruptive and annoying to all of us in the class. So please make sure your cell phone is silent before entering the class. Late work: Students may take any missed exam by the consent of the department members. And students are expected to provide their evidence for missing exam since 5 days after the onset of the exam. Disclaimer: This syllabus represent a best plan for the course, but, as with most plans, it is subject to changes made necessary by time, space and personal constraints as the course progresses. REQUIRED REFERENCES 1. Atlas, R.M Principles of Microbiology, WCB/Mc Graw Hill 2. Niclin, J., Graeme-Cook, K., Paget, T., and Killington, R Instant Notes in Microbiology. Bios Scientific Publishers Ltd. U.K. 3. Pelzar, M.J.; Chan, E.C.S., Kreig, N.R Microbiology (5 th Edition). Mc Graw Hill Pub. Co., New Delhi 4. Rangaswami, G. and Bhagyaraj, D.J Agricultural Microbiology (2 nd Edition). Asia Publishing House, New Delhi, India 5. Ross, F.C Introductory Microbiology. Charles E. Merrill Publishing Company 6. Subba Rao, N.S Soil Microbiology. (4 th Edition) Oxford & IBM Pub. Co. Pvt. Ltd., New Delhi 7. Subba Rao, N.S Advances in Agricultural Microbiology. (2 th Edition) Oxford & IBM Pub. Co. Pvt. Ltd., New Delhi 8. Tate, R.L Soil Microbiology (2 nd Edition). John Wiley and Sons. 5 P a g e Compiled by: Derib A.

6 1. INTRODUCTION 1.1. Definition and Scope of Microbiology Microbiology is the specific branch of biology that essentially deals with the elaborated investigation of microscopic organisms termed as microbes. These are typically microscopic organisms that are distributed abundantly both in the living bodies of plants and animals and also in the air, water and soil. Microbiology is the - scientific study of the microorganisms. In fact, microorganism invariably refers to the minute living body not detectable to the naked eyes (generally cannot be seen without magnifications). They are usually less than 1mm in size some of them are useful and some of them are harmful to human being. They are unicellular and one cell is capable of performing all the functions. Some microorganisms are multicellular with little or no cellular differentiation. Examples of microorganisms are Fungi, Bacteria, Virus, Protozoa and Algae. Interestingly, each and every microbe essentially bear both specific and special characteristic features that enable it to survive adequately in a wide spectrum of environments, such as: streams, ponds, lakes, rivers, oceans, ice, water-borne pipes, hot-springs, gastro-intestinal tract (GIT), roots of plants, and even in oil wells. In general, the microorganisms are usually characterized by very typical and extremely high degree of adaptability. Microbes are invariably distributed over the entire biosphere, lithosphere, hydrosphere, and above all the atmosphere. It is mainly concerned with a variety of vital and important aspects, such as: typical form, inherent structure, reproduction, physiological characteristics, metabolic pathways ( viz., anabolism, and catabolism), and logical classification. Besides, it includes the study of their: Distribution in nature, Relationship to each other and to other living organisms, Specific effects on humans, plants, and animals, and Reactions to various physical and chemical agents. 1 P a g e Compiled by: Derib A.

7 Disciplines of Microbiology The entire domain of microbiology may be judiciously sub-divided into a plethora of diversified, well-recognized, and broadly accepted fields. Basic disciplines Algology or Phycology: - study of algae; simple aquatic organisms ranging from single-celled forms to large seaweeds. Bacteriology: - Study of bacteria; the smallest, simplest, single-celled prokaryotic microorganisms and archaea prokaryotic microorganisms which constitute an ancient group intermediate between the bacteria and eukaryotes. Mycology: - Study of fungi; microscopic eukaryotic forms (molds and yeasts), higher forms (mushrooms, toadstools and puffballs), and slime molds. Protozoology: - Study of protozoans; animal like and mostly single-celled, eukaryotic organisms. Virology: - Study of viruses (infectious agents containing either DNA or RNA that require living cells for their replication/ or reproduction) and viral diseases. Parasitology: - Study of parasitism and parasites that include pathogenic protozoa, helminth worms and some insects. Microbial Ecology: - Study of interrelationships between microbes and environment. Microbial Morphology: - Study of detailed structures of microorganisms. Microbial Systematics: - Classification, naming, and identification of microorganisms and constructions of the phylogenetic tree of life. Microbial Physiology: - Metabolism of microbes at the cellular and molecular levels. Microbial Biochemistry: - Study of discovery of microbial enzymes and the chemical reactions carried out by them. Molecular Microbiology: - Study of genome (i.e., genomics) of microorganisms and construction of phylogenetic tree based on rrna. Microbial Genetics: - Study of heredity and variation in varieties. Molecular Biology: - The advanced study of the genetic material (DNA, RNA) and protein synthesis. 2 P a g e Compiled by: Derib A.

8 Applied disciplines Immunology: The immune system that protects against infections and attempts to understand the many phenomena that is responsible for both acquired and innate immunity, in addition to the study of antibody-antigen reactions in the laboratory. Agricultural Microbiology: Study of relationships of microbes and crops with an emphasis on control of plant diseases and improvement of yields. Food Microbiology: Interaction of microorganisms and food in relation to food bio-processing, food spoilage, food borne diseases and their prevention. Dairy Microbiology: Production of and maintenance in quality control of dairy products. Industrial Microbiology: Industrial uses of microbes in the production of alcoholic beverages, vitamins, amino acids, enzymes, antibiotics and other drugs. Marine Microbiology: Study of microorganisms and their activity concerning human and animal health in fresh, estuarine and marine waters. Air Microbiology: Role of aerospora in contamination and spoilage of food and dissemination of plant and animal diseases through air. Exomicrobiology: Exploration for microbial life in outer space. Diagnostic Microbiology: Fundamental principles and techniques involved in the study of pathogenic organisms as well as their application in the diagnosis of infectious diseases. Epidemiology and Public Monitoring: control and spread of diseases in communities. Biotechnology: The scientific manipulation of living organisms, especially at the molecular and genetic level to produce useful products Discovery and Historical Development of Microbiology Discovery of Microbiology Robert Hooke (1665) is the first person to report seeing microbes under the microscope. He is working with a crude compound microscope and he saw the cellular structure of plants around. He also saw fungi which he drew. However, because his lenses were of poor quality he was apparently unable to "see" bacteria. Anton van Leeuwenhoek (1676) for the first time he discovers the compound microscope and introduces to human being to a new world of microorganism through it. Also he described a number of microorganisms and made their drawings. French scientist J.J Thernanard (1803) 3 P a g e Compiled by: Derib A.

9 was the first person to suggest the role of microorganisms in fermentation of foods. Historical Development of Microbiology History of microbiology started when first microscope was made. Leeuwenhoek ( ) was a tailor. He used magnifying lens to examine fabrics for making dresses. To facilitate examining of cloth piece, he made a microscope whose magnification was only 30 times then he further made a large number of microscope improving each time. He examined drops of water, leaves, bloods and other such things with his microscope. He was fascinated to see microorganism in water, which he could never see without magnification. He saw bacteria, Protozoa, Stomata, and cells of Algae made their drawing and communicated to the royal society of London about different microorganism occurring in nature. Lous Pasteur ( ) using microscope found that good wine had red budding yeast cells, while sour wine had rod shaped bacteria. The yeast produce alcohol, while rod shaped bacteria produce lactic acid and caused sourness in the wine. He introduces the concept of pasteurilization (partial sterilization), in which grape juice is heated to F for minutes. These heat treatments kill undesirable microorganisms and control the sourness of wine. These days milk, butter, juices etc usually pasteurized in factor to increase their shelf life. Pasteur is called father of microbiology because of his enormous contribution. Pasteurization is not the same as sterilization. Pasteurization only kills organisms that may spoil the product, but it allows many microbes to survive, whereas sterilization kills all the living organisms in the treated material. 4 P a g e Compiled by: Derib A.

10 Table 1: Some early observations in microbiology before the dawn of golden era Time period Investigator Observations 4 th Century BC Aristotle Living things do not need parents, spontaneous generation apparently occurs Mid 1500s Fracastoro Contagion passes among individuals, objects and air. Mid 1600s Kircher Microscopic worms are present in blood of plague victims Mid 1600s Francisco Redi Fly larvae arise by spontaneous generation Late 1600s Van Leeuwenhoek Microscopic organisms are present in numerous environments Early 1700s Christian Fabricius Fungi cause rust and smut diseases in plants Early 1700s Joblot Existence of various forms of protozoa. Mid 1700s John Needham Microorganisms in broth arise by spontaneous generation Mid 1700s Lazzaro Heat destroys microorganisms in broth. Spallanzani Late 1700s Edward Jenner Recoverers from cowpox do not contract smallpox Mid 1800s Ignaz Semmelweis Chlorine disinfection prevents disease spread Mid 1800s John Snow Water is involved in disease transmission th C Golden Era 1848 Louis Pasteur the roles of microorganisms in fermentation, Swan-Neck Flask experiment Ferdinand Cohn discovered that bacteria multiply by dividing into two cells, and observed resistant structure called endospore in the cell 1928 Discovered Alexander Fleming Penicillium notatum, the fungus that kills staphylococcus aureus, a microorganism that is a leading cause of infection. 5 P a g e Compiled by: Derib A.

11 Theories on the origin of life Theory of spontaneous generation:- This theory was proposed in early day. According to it living thing organisms could arise spontaneously from non living matter. This theory was popular in those days, but later on some people did not agree with it. A number of experiments were conducted by different time to verify this theory scientifically. Finally Lous Pasteur ( ) scientifically proved that this theory was wrong. He showed that microorganisms were produced by the same kind of microorganism were responsible for fermentation. He establishes germ theory. never from non-living things. According to which living organisms arise from only from living things and The Theory of Biogenesis:- The issue was still unresolved in 1858, when the German scientist, Rudolf Virchow challenged the case for spontaneous generation with the concept of biogenesis, the claim that living cells can arise only from preexisting living cells. Arguments about spontaneous generation continued until 1861, when the issue was resolved by the French scientist Louis Pasteur. With a series of ingenious and persuasive experiments, Pasteur demonstrated that microorganisms are present in the air and can contaminate sterile solutions, but that air itself does not create microbes. He filled several shortly-necked flasks with beef broth and then boiled their contents. Some were then left open and allowed to cool. In a few days, these flasks were found to be contaminated with microbes. The other flasks, sealed after boiling, were free of microorganisms. From these results, Pasteur reasoned that microbes in the air were the agents responsible for contaminating nonliving matter such as the broths in Needham's flasks. Pasteur next placed broth in open-ended, long-necked flasks and bent the necks into S-shaped curves. The contents of these flasks were then boiled and cooled. The broth in the flasks did not decay and showed no signs of life, even after months. His unique design allowed air to pass into the flask, but the curved neck trapped any airborne microorganisms that might contaminate the broth. Pasteur showed that microorganisms can be present in nonliving matter-on solids, in liquids, and in the air. Furthermore, he demonstrated conclusively that microbial life can be destroyed by heat 6 P a g e Compiled by: Derib A.

12 and that methods can be devised to block the access of airborne microorganisms to nutrient environments. These discoveries form the basis of aseptic techniques, techniques that prevent contamination by unwanted microorganisms, which are now the standard practice in many laboratory and medical procedures. Modern aseptic techniques are among the first and most important things that a beginning microbiologist learns. Pasteur's work provided evidence that microorganisms cannot originate from mystical forces present in nonliving materials. Rather, any appearance of "spontaneous" life in non-living solutions can be attributed to microorganisms that were already present in the air or in the fluids themselves. Scientists now believe that a form of spontaneous generation probably did occur on the primitive earth when life first began, but they agree that this does not happen under today's environmental conditions The germ theory of disease (Koch's Postulates) In the early day man used to think that diseases are caused by some super natural powers. Involvement of microorganisms in disease was not known. Girolamo (1546) was working on contagious disease and treatment. He postulated that these diseases were transferred by germs (microorganism). Robert Koch ( ) is a bacteriologist. Became interested in anthrax and tuberculosis and suggested a method of confirming whether a microorganism cause a particular disease or not. Subsequently microorganisms were found to causes a number of disease in human being like typhoid (caused by bacteria Salmonella typhi), malaria (plasimodium spp), and tuberculosis (Mycobacterium spp). Polio (virus) and small box (pox virus). Today we know that microorganism causing most of human diseases. His procedure for defining the agent of any disease, called KOCH'S POSTULATES, consists of the following 4 steps: The microorganism must be present in the diseased animal and not present in the healthy animal; Cultivate the microorganism away from the animal in a pure culture; Symptoms of the disease should appear in the healthy animal after the healthy animal is inoculated with the culture of the microorganism and Isolate the microorganism from the newly infected animal and culture it in the laboratory. The new culture should be the same as the microorganism that was cultivated from the original diseased animal. 7 P a g e Compiled by: Derib A.

13 Figure 1. The diagrammatic representation of the Koch s criteria for proving that a specific microorganism causes a specific disease, i.e., the Koch s postulate Culture media and development of pure culture Culture media and its preparation Culture Media- is a liquid or solid nutrient medium prepared in the laboratory for the growth of microorganisms. The can be formulated from scratch or made by rehydrating commercially available powdered media. Media are constantly being developed for the use of identification and isolation of bacteria in the research of food, water, and microbiology studies. Medias in microbiology lab ranges from general purpose growth media to specifically differential media used in identification of microbe. Media supplies carbon, and nitrogen in a variety of forms. Microorganisms that grow in size and number on a culture medium are referred to as a culture. In order to use a culture medium must be sterile, meaning that it contains no 8 P a g e Compiled by: Derib A.

14 living organisms. We must have the proper nutrients, ph, moisture, and oxygen levels (or no oxygen) for a specific microorganism to grow. The most popular and widely used medium used in microbiology laboratories is the solidifying agent agar. Agar is a complex polysaccharide derived from red algae. Agar media are usually contained in test tubes or Petri dishes. The test tubes are held at a slant and are allowed to solidify on an angle, called a slant. A slant increases the surface area for organism growth. When they solidify in a vertical tube it is called a deep. The shallow dishes with lids to prevent contamination are called Petri dishes. Petri dishes are named after their inventor, Julius Petri, who in 1887 first poured agar into glass dishes. Types of Culture Media Culture media can be classified based on a number of criteria such as physical states, functional properties, chemical composition etc. Culture media classified based on physical States A. Liquid Media (Broth): These media do not contain any solidifying agent. Used for the propagation of large numbers of organisms, fermentation studies, and various other tests. Examples: nutrient broth, citrate broth, glucose broth, litmus milk, etc. B. Solid Media: Contain solidifying agent such agar, silica gel or gelatine. Nutrient agars, blood agar are examples of solid media that are used for developing surface colony growth of bacteria and molds. The development of colonies on the surface of a medium is essential when trying to isolate organisms from mixed cultures. C. Semisolid Media: Fall in between liquid and solid media. Although they are similar to solid media in that they contain solidifying agents such as agar and gelatine, they are more jellylike due to lower percentages of these solidifiers. These media are particularly useful in determining whether certain bacteria are motile or not. Culture media classification based on chemical compositions i. Synthetic media: type of media in which all the constituents are chemically defined. It is used to study specific nutritional requirements of microorganisms. E.g. Nutrient agar. ii. Complex media: a type of media in which there is one or more nutrients that is chemically undefined. Usually extracted from biological materials e.g. Beef extract, 9 P a g e Compiled by: Derib A.

15 peptone (casein) extract, yeast extract. Complex media satisfy the growth of most microorganisms. iii. Natural media: Naturally available and used as growth media without modification. E.g. milk Culture media classification based on their functional properties A. General Purpose Media: Media that support the growth of many microorganisms. B. Differential Media: They are media that are used to differentiate a specific microorganism from a given mixed population based on their biological characteristics. The purpose of a differential medium is to distinguish the target organism readily from any other organisms on the plate. It may contain dyes to be taken up or nutrients, which induce pigment formation or other special qualities so that the colonies are prominent. For example, on Miller-Schroth medium, Pseudomonas will sometimes grow, but colonies are green, while the target Erwinia colonies are bright orange-red. C. Selective Media: A culture media design to suppress the growth of unwanted microorganisms and encourage the growth of the desired microorganisms. The goal of a selective medium is to isolate only the target organism. Such media may contain antibiotics, fungicides, or other compounds that inhibit the growth of unwanted organisms ( exclusion). They also may contain nutrients that favor growth of the target organism ( enhancement). E.g. Bismuth sulfite agar inhibits all gram positive bacteria and gram negative bacteria except salmonella. D. Enrichment Media: A media used for preliminarily isolation that favours the growth of a particular microorganism. Unlike the above types of media, this medium may involve chemical, physiological, nutritional and environmental factors. It is used to enrich the required microbe. Development of pure culture A pure culture is sometimes called an axenic culture and refers to the growth of single type of microbe in an environment free of any other kind of living thing. Many microbiologists prefer to use the term axenic in order to avoid the misunderstanding that pure cultures are genetically pure. A pure, or axenic, culture is not genetically pure. It is a population of microbes of the same species but may contain some individual with mutations. 10 P a g e Compiled by: Derib A.

16 Pure cultures are used in the laboratory to study several characteristics that identify and classify microbes. Obtain pure cultures for identification is one of the most important laboratory procedures in agricultural microbiology. When examining diseased plant specimens, more often than not you will find several microorganisms, probably both fungi and bacteria, in the tissues. When you attempt to isolate, several different organisms may grown in your plates. Mostly it is not easy to tell which organisms is the cause of the disease under study. You have to make pure cultures of these organisms for any further investigation that could lead to the identification of the causative organism Solidifying agent: Agar Agar is used to solidify nutrient media for growing bacteria and fungi, it becomes liquid at 100 C and sets as solid solidify (into a firm gel). at 40 C. Agar powder will only dissolve in boiling water, once dissolved; the solution will remain liquid. Agar can be used in Petri dishes (plates) or in bottles (slopes). Agar can be obtained either as pure agar powder for adding to nutrient solutions which are prepared in the laboratory or ready mixed as a nutritive substrate in powder form with nutrients added according to specific recipes. For example, Sabouraud dextrose agar (SDA) and Malt extract agar (MEA) can be bought ready prepared or can easily be made up in the laboratory using pure agar powder plus the raw ingredients. N.B. Ready mixed agar products are generally more expensive than purchasing the pure agar powder and the media components separately. 11 P a g e Compiled by: Derib A.

17 2. BASIC CONCEPTS IN MICROSCOPY Microscope is material which is used to study microbes. The basic principle of microscope is 1. Microscopes are responsible for formation of image and play a central role in determining the quality of images that the microscope is capable of producing 2. To magnify particular specimen under which fine specimen detail can be observed in the microscope 2.1.Types of Microscopy Microscopes used in clinical practice are light microscopes. They are called light microscopes because they use a beam of light to view specimens. A. Compound light microscope:- is the most common microscope used in microbiology. It consists of two lens systems (combination of lenses) to magnify the image. Each lens has a different magnifying power. A compound light microscope with a single eye-piece is called monocular; one with two eye-pieces is said to be binocular B. Electron microscopes:- is a beam of electrons (instead of a beam of light) and electromagnets (instead of glass lenses) for focusing are called electron microscopes. These microscopes provide a higher magnification and are used for observing extremely small microorganisms such as viruses. Sub classification of microscope is based on nature of field i. Light microscopy (bright field microscopy) This is the commonly used type of microscope. In bright field microscopy the field of view is brightly lit so that organisms and other structures are visible against it because of their different densities. It is mainly used with stained preparations. Differential staining may be used depending on the properties of different structures and organisms. ii. Dark field microscopy In dark field microscopy the field of view is dark and the organisms are illuminated. A special condenser is used which causes light to reflect from the specimen at an angle. It is used for observing bacteria such as treponemes (which cause syphilis) and leptospires (which cause leptospirosis). 12 P a g e Compiled by: Derib A.

18 iii. Phase-contrast microscopy Phase-contrast microscopy allows the examination of live unstained organisms. For phase-contrast microscopy, special condensers and objectives are used. These alter the phase relationships of the light passing through the object and that passing around it. iv. Fluorescence microscopy In fluorescence microscopy specimens are stained with fluorochromes/ fluorochrome complexes. Light of high energy or short wavelengths (from halogen lamps or mercury vapour lamps) is then used to excite molecules within the specimen or dye molecules attached to it. These excited molecules emit light of different wavelengths, often of brilliant colours. Auramine differential staining for acid-fast bacilli is one application of the technique; rapid diagnostic kits have been developed using fluorescent antibodies for identifying many pathogens. Parts of microscope and its function 1. Tube:- Reflects light up to the viewer s eye 2. Rotating Objects:- Allows for quick change of objectives 3. Low Power Objective:-The first lens you use when doing proper microscope work. Usually 4 X 4. Medium Power Objective:-The second lens you use when doing proper microscope work. Usually 10 X 5. High Power Objective:-The highest magnification used. Usually 43 X. NEVER use the course adjustment when using this lens. 6. Stage Clips:- Use to keep the slide in place 7. Diaphragm:-Use to vary the amount of light passing through the slide. Usually it is better if the amount of light is low. 8. Light Source:- Sends light up through the diaphragm and through the slide for viewing 9. Eye Piece:-The part you look at with your eye. Usually 10 X magnification. 10. Neck :- Used to safely transport microscope 11. Stage :- Slides are placed on this 12. Coarse Adjustment:-Used to make large changes in focus. NOTE Never use this when viewing on high power 13. Fine Adjustment :- Used to small adjustments of focus 14. Base :- Used to safely transport the microscope 13 P a g e Compiled by: Derib A.

19 Fig 1. Parts of Microscope 14 P a g e Compiled by: Derib A.

20 2.2.Preparation and Fixation for Light Microscopic Examination Although living microorganisms can be directly examined with the light microscope, they often must be fixed and stained to increase visibility, accentuate specific morphological features, and preserve them for future study. Microscopic Techniques: Dyes and Staining Successful microscopy requires a proper specimen preparation (wet mount, smearing) and staining (various). Microscopes are of little use unless the specimens for viewing are prepared properly. Although resolution and magnification are important in microscopy, the degree of contrast between structures to be observed and their backgrounds is equally important. Nothing can be seen without contrast, so special techniques have been developed to enhance contrast. In addition, many living microorganisms are nearly transparent and often move rapidly about the slide. Consequently, cells are frequently immobilized and stained with dyes. Many different dyes and staining procedures, which have specific purpose and application, can be used in microscopy. Dyes or Stains The presence of colour gives the cells significant contrast so are much more visible as a consequence. A stain is a substance that adheres to a cell giving the cell colour. Microbiological stains are called dyes. A dye is an organic compound containing a chromophore group, which imparts colour to the compound, and an auxochrome, which is a dissociable group (one that give up or takes a proton to possess a negative or positive electrical charge) that binds to the substrate and intensifies the colour. Different stains have different affinities for different organisms or different parts of organisms. Dyes thus may be used to differentiate different types of organisms or to view specific parts of organisms. Basic dyes, which carry a positive charge, are more commonly used for staining than are negatively charged acidic dyes. Because microbes are known to possess a negative electric charge on them and get attracted to the positively charged ions of the stain. The most commonly used basic dyes are methylene blue, crystal violet, safrinine, and malachite green. Simple staining employs one of these basic dyes to stain the cells. Acidic dyes are sometimes used to 15 P a g e Compiled by: Derib A.

21 stain backgrounds, against which colourless cells can be seen, a technique called negative staining. To stain microorganisms, a drop of a liquid containing a microbe is placed on a glass microscope slide and the spread specimen forms a film (smear) and allowed to air dry. Then the organism is attached (fixed) to the slide, usually by passing the slide over a flame. Dye is then applied and washed off with water or other chemicals and allowed to dry to examine it with microscope. Wet mount: A wet mount is a non-dried specimen, typically a drop of specimen-containing medium. Wet mounts do not provide good contrast (i.e. it is difficult to see the microorganism) when using bright-field microscopy. Smears: It is a small volume of specimen containing medium that is spread (smeared) onto a microscope slide. A smear is the film obtained by placing a drop of a liquid containing a microbe on a glass microscope slide and allowing it to air dry. If you make smears too thick, you will have trouble seeing individual cells; if you make them too thin, you may find no organisms. If you stir the drop of medium too much as you spread it on the slide, you will disrupt cell arrangements. Fixing: A method of attaching the smeared organism to the slide is called fixing. Heat fixing is the most common form used. Heat fixation accomplishes three things: (1) It kills the organisms; (2) It causes the organisms to adhere to the slide; (3) It alters the organisms so that they more readily accept stains (dyes). If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed. If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps. Any cells remaining alive will stain poorly. If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains. Certain structures, such as capsules found on some microbes, are destroyed by heat-fixing so this step is omitted and these microbes are affixed to the slide just by air-drying. Staining Techniques A) Simple staining Simple staining is a staining procedure that uses only a single dye to stain the cell to increase the 16 P a g e Compiled by: Derib A.

22 contrast between colourless cells and a transparent background. It typically is only a single staining step and everything that stains is stained the same colour. It does not differentiate between different types of organisms. B) Differential staining Most stains used in microbiology are differential stains. Differential staining is a staining procedure that uses more than one dye to distinguish one group of organisms from another or to distinguish differences in the chemical composition of the cell of a microorganism. The two most frequently used differential staining techniques are the Gram stain and the acid fast stain. Gram stain The Gram staining technique, named after the Danish bacteriologist who originally devised it in 1844, Hans Christian Gram, is one of the most important staining techniques in microbiology. Gram staining procedure is usually the first to be performed to separate the bacteria into two major groups, the Gram-positive and Gram-negative. These two groups differ significantly in the chemical and physical structure of their cell wall. The Gram stain procedure involves four basic steps: 1. The smear is first flooded with the primary stain (crystal violet). The primary stain is the first dye applied in any multistep staining procedure and generally stains all of the cells. 2. The Gram s iodine is subsequently added as a mordant ( a chemical used to fix the staining reaction) to form the crystal violet-iodine complex, thereby decreasing the solubility of the dye within the cell. This step is commonly referred to as fixing the dye % alcohol or a mixture of alcohol and acetone is added as a solvent to act as a decolourizing agent. It readily removes the crystal violet-iodine complex from Gram-negative, but not Gram-positive, bacteria. A subsequent treatment with a decolourizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker Gram-positive cell walls, closing the pores as the cell wall shrinks during dehydration. As a result, the diffusion of the violet-iodine complex is blocked, and the bacteria remain stained. The length of the decolourization is critical in differentiating the gram-positive bacteria from the gram-negative bacteria. A prolonged exposure 17 P a g e Compiled by: Derib A.

23 to the decolourizing agent will remove all the stain from both types of bacteria. Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). 4. Finally, a counter stain is applied to impart a contrasting colour to the now colourless Gram-negative bacteria. The applied counter stain (either bas ic fuchsin or safrinin) then colourized the Gram-negative bacteria to pink. Fig 2. Steps in Gram stain procedure The structure of the thinner cell walls of Gram-negative bacteria cannot hold the dyes previously used once the decolourizer is applied. Note that the Gram-positive bacteria retain the stain (crystal violet-iodine complex) while the Gram-negative cannot retain this stain. The microorganisms that do not retain the crystal violet-iodine complex appear purple brown under microscopic examination. Bacteria that are stained by the Gram's method are commonly classified as Gram-positive or Gram non-negative. Others that are not stained by crystal violet-iodine complex are referred to as Gram-negative. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than Gram-negative bacteria. 18 P a g e Compiled by: Derib A.

24 3. TAXONOMY OF MICROBES Scientists carefully observe microorganisms and classify them into groups based on similar characteristics. In this chapter, you ll learn how to use scientific techniques to organize microorganisms into standard classifications based on a microorganism s characteristics. Organisms have traits that are similar to and different from other organisms. Scientists organize organisms into groups by developing taxonomy. Taxonomy is the science of classification of organisms based on a presumed natural relationship? Organisms that have similar characteristics are presumed to have a natural relationship and therefore are placed in the same group. Classification tries to show this natural relationship. Taxonomy has three components: Classification-the arrangement of organisms into groups based on similar characteristics, evolutionary similarity or common ancestry. These groups are also called taxa. Nomenclature- the act of given names to each organism. Each name must be unique and should depict the dominant characteristic of the organism. Identification- the process of observing and classifying organisms into a standard group that is recognized throughout the biological community Eukaryotic Microbes Eukaryotes are generally multi-cellular and are the organisms you are probably more familiar with, including plants, animals, algae, fungi and protists (protozoa). Algae are relatively simple plants; their size varies from 1 μm to several feet, they contain the green pigment chlorophyll, so can carry out photosynthesis (autotrophic) and are found most commonly in aquatic environments or in damp soil. They cause problems by clogging (block) water pipes, releasing toxic chemicals into water bodies, or growing in swimming pools but extracts of some species have commercial uses: as emulsifiers for foods such as ice-creams; as a source of agar used as solidifying agent in microbial medias and as anti-inflammatory drugs for ulcer treatment. Fungi - are either saprophytes or parasites. They have eukaryotic cell structures which, like algae, have rigid cell walls. They form characteristic hyphae called mycelium which may be septate, nonseptate or coenocytic. They form fruiting structures called conidia or exospores and 19 P a g e Compiled by: Derib A.

25 endospores. Spores of fungi are always present in air, dust and soil. Multicellular fungi are also called molds while yeast is an important unicellular fungus. Size range of molds is μm and yeast has size varying in the range of 5-10 μm. Molds have considerable value; they are used to produce antibiotics- penicillin, cephalosporin etc, fermented products like soy sauce, tempeh, miso, Roquefort and Camembert cheeses, and many other products. But they are also implicated in various human, animal and plant diseases including athlete s foot and the moldy spoilage of grains and peanuts. The unicellular yeasts are widely used in Baking industry and for the production of all alcoholic beverages like wine, beer etc. On the other hand, some yeasts cause food spoilage and diseases such as vaginitis and thrush (an oral infection). Protozoa are unicellular eukaryotic organisms (animals), which are motile having cilia, flagella and pseudopodia, they are either saprophytic or parasitic or parasitic. They are generally present in soil, water and marshy places and their size varies from μm. They are differentiated on the basis of morphological, nutritional and physiological characteristics. Their role in nature is varied, but the best known protozoa are the few that cause disease in human beings and animals, such as malaria in humans. Some protozoa are beneficial, such as those found in stomach of cattle, sheep and termites that help digest food Prokaryotic Microbes Prokaryotes, organisms with cells that lack membrane-bound organelles, and are unicellular, which are contained in the domains of bacteria, archaea and viruses. Bacteria are unicellular microorganisms. Their size varies from μm and has various shapes, such as: rod, coccus or spiral shape. They have prokaryotic cellular organization and cell division is usually by binary fission. Some bacteria having mycelia morphology are known as Actinomycetes and are very important in production of antibiotics. Bacteria are important in agriculture and play important role in cycle of biological nitrogen fixation. Archaea are procaryotes that are distinguished from Bacteria by many features, most notably their unique ribosomal RNA sequences. They also lack peptidoglycan in their cell walls and have unique membrane lipids. Some have unusual metabolic characteristics, such as the methanogens, which generate methane gas. Many archaea are found in extreme environments. Pathogenic 20 P a g e Compiled by: Derib A.

26 archaea have not yet been identified. Domain Eucarya includes microorganisms classified as protists or Fungi. Animals and plants are also placed in this domain. Viruses are become identified with the discovery of electronic microscope in The first virus to be seen and identified by use of electronic microscope is known as Tobacco masaic appearing as a regular rod shaped particle measuring about 25 X 300 nanometer (about 1/1000 of micrometer. The size of virus can be determined from: the pore size that it can be filtered and the speed of centrifugation and sedimentation characteristics. Viruses have two distinctive patterns of shape, namely spherical or helical. The proteins that make up viruses structural parts particle is called structural proteins. Among these are found: protein coat covering their entire cell called a capsid. Capsids have two functions, facilitating entry to host cells, and protect the delicate viral nucleic acid. Table 1. A comparison of eukaryotic and prokaryotic characteristics and structures Characteristics or structures Prokaryotes Eukaryotes Size (in diameter) µm 5 50 µm Cell division Chromosome replication followed by Mitosis followed by division binary fission (No mitosis and meiosis) (meiosis) Chromosome location Located in a region of the cell called nucleoid, which is not membrane bound Contained within the membrane bound nucleus Nucleus Absent, DNA resides as an irregular mass Present; DNA is form of forming the nucleoid region chromosomes enclosed in nucleus with nuclear membrane Chromosome/D NA Single, circular, no histones Multiple, linear, DNA wrapped around histones Cell wall Complex cell wall composed of Present in most algae and fungi; peptidoglycan except in mycoplasmas absent in protozoa; absence of peptidoglycan layer Cell membrane Relatively symmetric Highly asymmetric Flagella Simple flagella composed of protein Complex flagella: made up of a 21 P a g e Compiled by: Derib A.

27 subunits 9+2 arrangement of microtubules Membrane-boun Absent Present: includes the nucleus, d organelles mitochondria, chloroplasts, endoplasmic reticulum, golgi apparatus, lysosomes, and peroxisomes Ribosome Small ribosomes with sedimentation Large ribosomes with coefficient of 70S sedimentation coefficient of 80S; mitochondria and chloroplasts have 70S ribosomes Glycocalyx Capsule or slime layer Possibly in those without cell wall Cytoplasmic Sterols absent except in mycoplasma Contain sterols membrane Cytoplasmic Absent Present streaming Cytoskeleton Absent Present Respiration Associated with cytoplasmic membrane Associated with organelle called mitochondria Photosynthesis Present; no chloroplast Present; possess chloroplast Motility Flagella, axial filament, and some gliders Flagella (cilia) or ameboid movement, and some gliders Reproduction Asexual (binary fission) conjugation rare, sporulation is for survival but not for Sexual or asexual; conjugation, sporulation reproduction Metabolism Faster Slower than prokaryotes Habitat Aerobic and anaerobic environments Almost exclusively aerobic environments 22 P a g e Compiled by: Derib A.

28 4. THE STRUCTURE AND FUNCTIONS OF PROKARYOTIC (BACTERIAL) CELLS 4.1. General Characteristics of Bacteria Bacteria (singular: bacterium) are a large domain of prokaryotic microorganisms which are hold the following general morphological characteristics. 1. Size: bacteria are the smallest of all most typically a few micrometers (1/1000mm) only very few spp approximately 0.5µm-1 µm in diameter. They have high surface area to volume ratio, so have efficient rate of material exchange, 2. Shape: have a wide range of shapes, ranging from spheres to rods and spirals. Cocci (coccus) = berry, = spherical shaped Baccilli (bacillus) = cylindrical or rod shaped Spiral or helical shaped Figure Bacterial shapes. Most bacteria are (a) rod shaped, (b) spherical or (c) curved. These basic shapes may join to form (d) pairs, (e and f) chains, (g) sheets, (h) packets or (i) irregular aggregates 3. Cell arrangement: when observed under microscope, bacteria usually exist attached other. While the spiral bacteria occur, as a single, most other species of bacteria grow in characteristic arrangement patterns. 23 P a g e Compiled by: Derib A.

29 4. Habitat: Present in most habitats on earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals, providing outstanding examples of mutualism in the digestive tracts of humans, termites and cockroaches. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a milliliter of fresh water; in all, there are approximately five nonillion ( ) bacteria on Earth, forming a biomass that exceeds that of all plants and animals. 5. Utility (application): Bacteria are vital in Recycling nutrients, with many steps in nutrient cycles depending on these organisms, such as the fixation of nitrogen from the atmosphere and putrefaction. In the biological communities surrounding hydrothermal vents and cold seeps, bacteria provide the nutrients needed to sustain life by converting dissolved compounds such as hydrogen supplied and methane. The vast majority of the bacteria in the body are rendered harmless by the protective effects of the immune system, and a few are beneficial. However, a few species of bacteria are pathogenic and cause infectious diseases, including cholera, syphilis, anthrax, leprosy, and bubonic plague. The most common fatal bacterial diseases are respiratory infections, with tuberculosis alone killing about 2 million people a year, mostly in sub-saharan Africa. In industry, bacteria are important in sewage treatment and the breakdown of oil spills, the production of cheese and yogurt through fermentation, the recovery of gold, palladium, copper and other metals in the mining sector, as well as in biotechnology, and the manufacture of antibiotics and other chemicals. 6. Most bacteria have not been characterized, and only about half of the phyla of bacteria have species that can be grown in the laboratory. 7. Cellular organization: unlike cells of animals and other eukaryotes, bacterial cells do not contain a nuclear membrane and other well compartmentalized organelles, rather they rarely harbor few of membrane-bound organelles. The scientific classification changed after the discovery in the 1990s that prokaryotes consist of two very different groups of organisms that evolved independently from an ancient common ancestor. These evolutionary domains are called Bacteria, belongs to kingdom monera and Archaea. 24 P a g e Compiled by: Derib A.

30 4.2. Bacterial Cell External structure In most bacteria a cell wall is present on the outside of the cytoplasmic membrane. A common bacterial cell wall material is peptidoglycan called murein in older sources, which is made from polysaccharide chains cross-linked by peptides containing D-amino acids. Bacterial cell walls are different from the cell walls of plants and fungi, which are made of cellulose and chitin, respectively. The cell wall of bacteria is also distinct from that of Archaea, which do not contain peptidoglycan. The cell wall is essential to the survival of many bacteria, and the antibiotic penicillin is able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan. There are broadly speaking two different types of cell wall in bacteria, called Gram-positive and Gram-negative. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria have a relatively have thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. Most bacteria have the Gram-negative cell wall. In many bacteria an S-layer of rigidly arrayed protein molecules covers the outside of the cell. \ This layer provides chemical and physical protection for the cell surface and can act as a macromolecular diffusion barrier. S-layers have diverse but mostly poorly understood functions, but are known to act as virulence factors in Campylobacter and contain surface enzymes in Bacillus stearothermophilus. Flagella are rigid protein structures, about 20 nanometres in diameter and up to 20 micrometres in length, which are used for motility. Flagella are driven by the energy released by the transfer of ions down an electrochemical gradient across the cell membrane. Fimbriae are fine filaments of protein, just 2 10 nanometres in diameter and up to several micrometers in length, distributed over the surface of the cell, (resemble fine hairs) are believed to 25 P a g e Compiled by: Derib A.

31 be involved in attachment to solid surfaces or to other cells and are essential for the virulence of some bacterial pathogens. Pili (sing. pilus) are cellular appendages, slightly larger than fimbriae, that can transfer genetic material between bacterial cells in a process called conjugation (see bacterial genetics, below) Capsules or slime layers are produced by many bacteria to surround their cells, and vary in structural complexity: ranging from a disorganized slime layer of extra-cellular polymer, to a highly structured capsule or glycocalyx. These structures can protect cells from engulfment by macrophages. They can also act as antigens and be involved in cell recognition, as well as aiding attachment to surfaces and the formation of biofilms Internal structure Cell (plasma) membrane is the cellular structure which separates (limits), the cy ctoplasmic contents from the external environment. Bacteria plasma membrane is the site of many biological functions that are accomplished by specialized internal organelles of other eukaryotes. It is specialized to perform such important functions as: selective transport of molecules into and out of cells respiration and photosynthesis secretion of extracellular enzymes regulation and replication Structure and composition of Plasma membrane Structurally: 20-30% =phospholipids (forming a bilayer in which proteins embedded), 50-70% =protein. Each of phospholipids has a charged polar head (soluble) group = hydrophilic and non-charged tail region (non-soluble) = hydrophobic. In comparison to eukaryotes, the intracellular features of the bacterial cell are extremely simple., i.e., bacteria do not contain organelles in the same sense as eukaryotes. Instead enclosed within the plasma membrane are found internal structures of the cell, occupied by a fluid portin called cytoplasmic area, mainly composed of? Ribosome densely packed organelles found throughout cytoplasm, protein factory. Cytoplasmic inclusions. The most prominent ones are 26 P a g e Compiled by: Derib A.

32 Nucleoid the nuclear area (material) consists of a single circular chromosome, the thread like body which carries hereditary information. Specialized groups of bacteria that contain more complex intracellular structures, some of which are discussed below. i. The bacterial chromosome and plasmids The bacterial chromosome is not packaged using histones to form chromatin as in eukaryotes but instead exists as a highly compact super coiled structure, the precise nature of which remains unclear. Most bacterial chromosomes are circular although some examples of linear chromosomes exist (e.g. Borrelia burgdorferi). Along with chromosomal DNA, most bacteria also contain small independent pieces of DNA called plasmids that not essential for growth and often encode for traits that are advantageous but not essential to their bacterial host. Plasmids can be easily gained or lost by a bacterium and can be transferred between bacteria as a form of horizontal gene transfer. ii. Ribosome (70S, where S=Svedberg units) iii. Intracellular membranes Groups of phototrophs, nitrifying and methane-oxidizing bacteria etc all have intracellular membranes. Intracellular membranes are also found in bacteria belonging to the poorly studied Planctomycetes group, although these membranes more closely resemble organellar membranes in eukaryotes and are currently of unknown function. iv. Cytoskeleton Cytoskeletal elements play essential roles in cell division, protection, shape determination, and polarity determination in various prokaryotes. v. Nutrient storage structures In order to accommodate excess (transient) levels of nutrients, bacteria contain several different methods of nutrient storage. Many bacteria store excess; Carbon in the form of polyhydroxyalkanoates or glycogen. Nitrate in vacuoles. Sulfur is most often stored as elemental (S 0 ) granules which can be deposited either intra- or extracellularly. Sulfur granules are especially common in bacteria that use hydrogen sulfide as an electron source. 27 P a g e Compiled by: Derib A.

33 vi. Gas vesicles Gas vesicles are spindle-shaped structures found in some planktonic bacteria that provide buoyancy to these cells by decreasing their overall cell density. vii. Carboxysomes Carboxysomes are intracellular structures found in many autotrophic bacteria such as Cyanobacteria, Knallgasbacteria, Nitroso- and Nitrobacteria. They are proteinaceous structures resembling phage heads in their morphology and contain the enzymes of carbon dioxide fixation in these organisms (especially ribulose bisphosphate carboxylase/oxygenase) viii. Magnetosomes Magnetosomes are intracellular organelles found in magnetotactic bacteria that allow them to sense and align themselves along a magnetic field (magnetotaxis). Magnetosomes are composed of the mineral magnetite or greigite and are surrounded by a lipid bilayer membrane. The morphology of Magnetosomes is species-specific. ix. Endospores Perhaps the most well-known bacterial adaptation to survival stress is highly resistant to many different types of chemical and an environmental stress is the formation of endospores. 28 P a g e Compiled by: Derib A.

34 5. MICROBIAL GROWTH AND NUTRITION 5.1. Microbial Growth Bacterial growth and reproduction Although widely varying in morphology, bacteria share one major characteristic: they divide by simple binary fission. This means that one cell grows to about double its original size and then splits into two genetically identical cells. Since DNA replication occurs before the cells divide, each new cell, called a daughter cell, gets a complete genome (a full set of genes). The two genetically identical daughter cells are called clones. All the progeny of a single original cell form a mass of cells on a solid surface such as agar is called a colony. If the original form was not a single cell, for example, it was a chain of cocci, that entire chain of cells and all its progeny will form a single colony. So a colony forming unit (CFU) may include the progeny of a single cell, or it may include the progeny of several cells that were originally connected to each other. Steps involved for bacteria reproduction 1. The cell elongates and chromosomal DNA is replicated. 2. The cell wall and cell membrane pinch inward and begin to divide. 3. The pinched parts of the cell wall meet, forming a cross wall completely around the divided DNA. 4. The cells separate into two individual daughter cells. Figure. Binary fission in bacteria 29 P a g e Compiled by: Derib A.

35 The growth curve Bacterial growth over time can be graphed as cell number versus time. This is called a growth curve. The cell number is plotted as the log of the cell number, since it is an exponential function. Regardless of the generation time, in a growing culture the plot of the log of cell number versus time gives a characteristic curve. This curve typically has four distinct phases: lag phase, exponential (log) phase, stationary phase, and death phase. In cells that have been freshly inoculated into a new growth medium, the lag phase is the first phase observed. It is characterized by no increase in cell number; however, the cells are actively metabolizing, in preparation for cell division. Depending on the growth medium, the lag phase may be short or very long. For example, if a culture in a rich growth medium that supplies most of the cells requirements is inoculated into a poor medium that requires the cells to make most of their own amino acids and vitamins, the lag phase will be very long. The cells must activate the metabolic pathways for amino acid and vitamin synthesis and must make enough of these nutrients to begin active growth. In contrast, cells that are simply diluted from one medium to a fresh tube of the same medium may show virtually no lag phase, since they need not change their metabolism. Once cells are actively metabolizing they begin DNA replication and shortly after that the cells divide. This begins the second phase of growth called the exponential or log phase of growth. This is the period in which the cells grow most rapidly, doubling at a fairly constant rate. The time it takes the culture to double is called the generation time. The generation time can be easily obtained from the exponential phase of a growth curve. The log of the cell number versus time will yield a straight line when the cells are in exponential growth. The generation time can be read directly from the graph using two points on the straight line that represent a two-fold increase in the cell number. Generation Time The generation time is the amount of time needed for a cell to divide. This varies among organisms and depends upon the environment they are in and the temperature of their environment. Some bacteria have a generation time of 24 hours, although the generation time of most bacteria is between 1 to 3 hours. Bacterial cells grow at an enormous rate. For example, 30 P a g e Compiled by: Derib A.

36 with binary fission, bacteria can double every 20 minutes. In 30 generations of bacteria (10 hours), the number could reach one billion. It is difficult to graph population changes of this magnitude using arithmetic numbers, so logarithmic scales are used to graph bacterial growth. Phases of Growth There are four basic phases of growth: the lag phase, the log phase, the stationary phase, and the death phase. 1. THE LAG PHASE: In the lag phase there is little or no cell division. This phase can last from one hour to several days. Here the microbial population is involved in intense metabolic activity involving DNA and enzyme synthesis. This is like a factory shutting down for two weeks in the summer for renovations. New equipment is replacing old and employees are working, but no product is being turned out. 2. THE LOG PHASE: In the log phase, cells begin to divide and enter a period of growth or logarithmic increase. This is the time when cells are the most active metabolically. This is the time when the product of the factory must be produced in an efficient matter. In this phase, however, microorganisms are very sensitive to adverse conditions of their environment. 3. THE STATIONARY PHASE: This phase is one of equilibrium. The growth rate slows, the number of dead microorganisms equals the number of new microorganisms, and the population stabilizes. The metabolic activities of individual cells that survive will slow down. The reason why the growth of the organisms stops is possibly that the nutrients have been used up, waste products have accumulated, and drastic harmful changes in the ph of the organisms environment have occurred. There is a device called a chemostat that drains off old, used medium and adds fresh medium. In this way a population can be kept in the growth phase indefinitely. 4. THE DEATH PHASE: Here the number of dead cells exceeds the number of new cells. This phase continues until the population is diminished or dies out entirely. 5. THE PROLONGED DECLINE PHASE: This phase is marked by a very gradual decrease in the number of viable cells in the population lasting for months to years. 31 P a g e Compiled by: Derib A.

37 Figure. The Growth Curve Measurements of microbial growth (Bacterial counts) Bacterial growth can be measured by both direct and indirect methods. It generally considered at two levels, that is growth in size and growth in numbers. Growth in numbers can be measured by bacterial counts. There are two types of bacterial counts. 1. Total count 2. Viable count Total count: this gives the total number of cells in the sample which includes the living and dead cells. It can be obtained by following methods. Direct counting under microscope By using culture counter Direct counting using stained smears prepared by spreading a known volume of culture over a measured area on the slide. By separating the cells by centrifugation or filtration and measuring their wet or dry weight. Viable count: it measures the number of living cells only, that is, the cells capable of multiplication. Viable counts are obtained by dilution or plating methods. Dilution method: In this method, the suspension is diluted to a point where there is growth when inoculated in to suitable liquid media. Several tubes are inoculated with varying dilutions and the viable count is measured statistically from the number of tubes showing growth. This method is mainly used for measuring the presumptive coliform count in drinking water. 32 P a g e Compiled by: Derib A.

38 Plating method: In this method, appropriate dilutions are inoculated on solid media. The numbers of colonies develop after incubation gives the estimate of viable count Nutrient Requirement The growth of any microbe depends on the physical environment and the available source of chemicals to use as nutrition. Microbes are incredibly diverse and every microbe has its own minimum physical environment and nutritional requirement. Each microbe can grow only if presented with the right nutrients/conditions. Major elements: are elements that are required in large amounts to make up the cell constituents. They are the essential components of proteins, carbohydrates, lipids and nucleic acids. It includes carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorous, potassium, magnesium, calcium, and iron. Trace elements: are elements that are required in small amount by all cells. These elements form parts of enzymes or may be required for enzyme function. It includes cobalt, zinc, copper, molybdenum and manganese. Growth factors: Some bacteria cannot synthesize some of their cell constituents, such as amino acids, vitamins, purines and pyrimidines, from the major elements. These organisms can only grow in environments where such compounds are available. Those low molecular weight compounds (amino acids, vitamins, purines and pyrimidines) that must be provided to a particular bacterium are called growth factors. The source of carbon distinguishes different groups of prokaryotes as autotrophs and heterotrophs. Autotrophs are microbes that use inorganic carbon in the form of carbon dioxide as a carbon source. Heterotrophs are microbes that use organic carbon as a carbon source. Based on the way of harvesting energy, prokaryotes are classified into two major groups; phototrophs and chemotrophs. Organisms that harvest the energy of sunlight are called phototrophs. Organisms that obtain energy by metabolizing chemical compounds are called chemotrophs. 33 P a g e Compiled by: Derib A.

39 Microbiologists often group prokaryotes according to the energy and carbon sources they utiilize as photoautotrophs (e.g. Cyanobacteria), chemoautotrophs or chemolithoautotrophs (e.g. Archaea), photoheterotrophs (e.g. some purple non-sulfur vacteria) and chemoorganoheterotrophs (most bacteria). Table 2: Energy and carbon sources used by different groups of prokaryotes 5.3. Environmental Requirement The growth of microbes depends not only on the provision of nutrients but also on the presence of appropriate environmental conditions such as temperature, ph, water activity, oxygen, pressure and radiation. Each species of microbes has well defined upper and lower limits of these factors within which it grows and outside of which growth stops. 34 P a g e Compiled by: Derib A.

40 Table 3: Terms used to describe microbes in relation to their requirement of environmental factors for optimum growth. 35 P a g e Compiled by: Derib A.

41 6. MICROBIOLOGY OF PLANT PATHOGENIC MICROORGANISMS 6.1. Causes of Plant Disease Plant diseases caused by pathogens. Hence a pathogen is always associated with a disease. In other way, disease is a symptom caused by the invasion of a pathogen that is able to survive, perpetuate and spread. Further, the word pathogen can be broadly defined as any agent or factor that incites pathos or disease in an organism or host Fungi Most plant pathogenic fungi form hyphae i.e. filamentous cells which extend by apical growth and an ordered system of branching. The network of hyphae which results from such growth is called a mycelium, and the interconnected hyphal network derived from one propagule is termed a colony. The apical mode of growth of most fungi is the key to the success of these organisms both as saprotrophs and parasites. Unlike unicellular organisms, filamentous fungi are able to extend through soil, plant litter or living tissues. As a hypha grows through the substrate it secretes extracellular enzymes which digest complex molecules. The products of this process are then absorbed by the hypha. As the nutrients become exhausted the hypha simply grows on to explore a new area Bacteria The majority of plant-pathogenic bacteria are unicellular, with cell division by binary fission; the subsequent activities of the separated cells are not coordinated in any way. Sometimes individual bacterial cells aggregate to form substantial colonies, as in crown gall infections. In other cases cells spread throughout an entire organ or physiological system. Growth of single-celled bacteria, like fungi, also involves the production of extracellular enzymes. Many plant pathogenic bacteria possess flagella and therefore motile and capable of moving along nutrient gradients and towards host signal molecules. 36 P a g e Compiled by: Derib A.

42 6.4. Viruses As first sight viruses might appear ill-equipped to act as pathogens, due to their extreme dependence on living cells. Virus parasitism is unique, in that the parasite is incorporated into the metabolism of the host cell. After gaining entry into a living cell, the nucleic acid component of the virus is released from its protein coat. The viral genome is then translated and replicated, and numerous new virus particles are assembled from the newly synthesized nucleic acid and protein. A virus can thus be visualized as a set of instructions for making more viruses, packaged in a protective coat. In contrast to fungi and bacteria, viruses do not attack the structural integrity of their host tissues. Example: Tobacco mosaic virus (TMV). 37 P a g e Compiled by: Derib A.

43 7. SOIL MICROORGANISMS (Group Assignments) 7.1. Influence Of Plants On Soil Microorganisms 7.2. Rhizosphere Environment 7.3. Factors Affecting the Rhizosphere 7.4. Plant Derived Compounds In the Rhizosphere 7.5. Organisms Inhabiting the Rhizosphere 7.6. Microbial Plant Interaction In the Rhizosphere 7.7. Plant Growth Promoting Rhizobacteria (PGPR) 38 P a g e Compiled by: Derib A.

44 8. MICROBIAL INTERACTION IN SOIL SYSTEM Microorganisms are ubiquitous in their occurrence and in natural environments they interact among themselves and with plants, animals, and their niches as a result different types of microbial interactions established. Interaction of organisms with each other and with their physical environment contributes to the functioning of ecosystems. The reasons for microbial interactions are the competition for nutrients (including oxygen) and space in an ecological niche. On the basis of relative advantage to each partner i.e. hosts and microorganisms, the relationships (interactions) are basically of three types. Neutralism: when the host remains unaffected by the microbes. Mutualism: when both partners get benefits from the association. Parasitism: when one partner gets benefits and the second partner suffers from damages Plant-Microbe Interaction The aboveground (foliage) and below ground (root) portions of plants are constantly interact with a large number of microorganisms (e.g. bacteria, actinomycetes, fungi, algae, nematodes, and viruses) and develop several types of interrelationships. Considering the results of interactions, the microbe may develop destructive, neutral, or beneficial associations with plants Aboveground microbe-plant interaction (P hyllosphere or Phylloplane) Microbial interactions on aboveground parts of plants occur in several ways where the foliage especially leaf surface (phyllosphere and phylloplane) acts as microbial niche. The association may be destructive (diseased) which i s caused by pathogenic microorganisms or beneficial (symbiosis). Some examples of microbial pathogens causing disease on phyllosphere include Pseudomonas pisi (stem blight of pea), Alternaria solani (early blight of potato), Puccinia tritici (rust of wheat), etc. The excellent example of beneficial association is the development of stem nodules by some legumes species like Neptumia when interact with Azorhizobium bacteria Below ground microbe-plant interaction (Rhizosphere or Rhizoplane) The rhizosphere is the zone of soil immediately adjacent to plant roots in which the kinds, numbers, or activities of microorganisms differ greatly from that of the bulk soil. The rhizosphere is today regarded as the zone of microbial proliferation in and around roots. A 39 P a g e Compiled by: Derib A.

45 variety of light and electron microscopic techniques have been used to observe bacteria and fungi around roots (the ectorhizosphere), on the root surface (the rhizoplane) and within the root (the endorhizosphere). Some examples of microbial pathogens causing disease on the rhizosphere include Streptomyces scabies (scab of potato), Rhizoctonia solani (root rot of potato and many plants), Meloidogyne incognita (root knots of tomato and many plants), etc. The beneficial associations (Symbiosis) as a result of in teraction of microorganisms with plant roots may or may not develop apparent symbiotic structure. Symbioses between two organisms vary based on; 1. Degree of intimacy: the degree of closeness, proximity of the association between interacting organisms. The degree of intimacy could be: Ectosymbiosis: in which one organism remains outside the other organism i.e. external to the cells and tissues of the partner. Endosymbiosis: in which one organism is found within the other organism. Ecto/endosymbiosis: in which one organism lives both on the inside and the outside of the other organism. 2. Relative advantage each partner gets from the association: some associations are equally beneficial to both partners and some are beneficial to only one of the organisms. Examples of symbiotic associations are: Mutualism: refers symbiotic associations in which both organisms are equally benefited. Commensalism: refers relationships between two organisms in which one is benefit from the association and the other is unaffected. Microorganism form symbiotic relationship for either of the following purposes: i. Protection against: extreme environment: (e.g. desiccation, temperature, ph, etc.), invasion by disease causing microorganisms, toxic products (e.g. breakdown and removal of toxic substance like urea and uric acid by bacteria in the excretory organs of insects). ii. Direct provision of nutrients (for instance, fungi in roots of plants increase water absorption capacity of roots and increase absorption of nutrients. iii. Indirect provision of one or more nutrients to its partner (e.g. protozoa and bacteria in cellulose digestion in ruminants). 40 P a g e Compiled by: Derib A.

46 Many microbes (bacteria, fungi, etc) have important symbioses with plant roots. The rhizosphre, which is a thin layer soil immediately adjacent to root hairs of plants typically, contains 10 9 microbes/g of soil. Many rhizosphere organisms are ectosymbionts and others are endosymbionts.. 41 P a g e Compiled by: Derib A.

47 9. ROLE OF MICROORGANISMS IN SOIL FERTILITY AND CROP PRODUCTION CYCLES 9.1. Microbial Metabolism Microbial metabolism is the means by which a microbe obtains the energy and nutrients (e.g. carbon) it needs to live and reproduce. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe s ecological niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles. Biogeochemical Cycling: Microorganisms, in the course of their growth and metabolism, interact with each other in the cycling of nutrients, including carbon, nitrogen, phosphorus, sulfur, iron, and manganese. This nutrient cycling, called biogeochemical cycling when applied to the environment, involves both biological and chemical processes and is of global importance. Nutrients are transformed and cycled, often by oxidation-reduction reactions that can change the chemical and physical characteristics of the nutrients. All of the biogeochemical cycles are linked and the metabolism-related transformations of these nutrients make life on Earth possible. Types of microbial metabolism All microbial metabolisms can be arranged according to the following principles: 1. How the organism obtains carbon for synthesizing cell mass: Autotrophic:- carbon is obtained from carbon dioxide (CO 2 ) Heterotrophic:- carbon is obtained from organic compounds Mixotrophic:- carbon is obtained from both organic compounds and by fixing carbon dioxide 2. How the organism obtains energy for living and growing: Chemotrophic:- energy is obtained from external chemical compounds Phototrophic:- energy is obtained from light Fermentation: Fermentation is a metabolic process whereby electrons released from nutrients are ultimately transferred to molecules obtained from the breakdown of those same nutrients (it is the enzymatic decomposition and utilization of foodstuffs, particularly carbohydrates, by microbes). 42 P a g e Compiled by: Derib A.

48 Fermentation in food processing typically is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic conditions. A more restricted definition of fermentation is the chemical conversion of sugars into ethanol. The science of fermentation is known as zymurgy. Fermentation usually implies that the action of microorganisms is desirable, and the process is used to produce alcoholic beverages such as wine, beer, and cider. Fermentation is also employed in the leavening of bread, and for preservation techniques to create lactic acid in sour foods such as sauerkraut, dry sausages, and yogurt, or vinegar (acetic acid) for use in pickling foods. Ethanol fermentation, the production of ethanol for use in food, alcoholic beverage, fuel and industry Industrial fermentation, the breakdown and re-assembly of biochemicals for industry, often in aerobic growth conditions Fermentative hydrogen production, the fermentative conversion of organic substrate to biohydrogen manifested by a diverse group of bacteria. Tea processing, the term used in the tea industry for the aerobic treatment of tea leaves to break down certain unwanted chemicals and modify others to develop the flavor of the tea. In general, fermentation is any anaerobic biochemical reactions in which an enzyme (or several enzymes produced by a microorganism) catalyses the conversion of one substance into another; especially the conversion (using yeast) of sugars to alcohol or acetic acid with the evolution of carbon dioxide. Uses of Fermentation: The primary benefit of fermentation is the conversion of sugars and other carbohydrates, e.g., converting juice into wine, grains into beer, carbohydrates into carbon dioxide to leaven bread, and sugars in vegetables into preservative organic acids. Food fermentation has been said to serve five main purposes: Enrichment of the diet through development of a diversity of flavors, aromas, and textures in food substrates Preservation of substantial amounts of food through lactic acid, alcohol, acetic acid and alkaline fermentations Biological enrichment of food substrates with protein, essential amino acids, essential fatty acids, and vitamins Elimination of antinutrients A decrease in cooking times and fuel requirements 43 P a g e Compiled by: Derib A.

49 9.2. Carbon Transformation and Soil Organic Matter Formation Carbon Cycle Carbon is the building block of life. Plants obtain carbon from atmospheric carbon dioxide (CO 2 ) through photosynthesis, during which the chloroplasts in the plant cells convert CO 2 carbohydrates. It is the cycling of carbon from the atmosphere through plants and algae, to animals and micro-organisms and back to the atmosphere that maintains earth's atmosphere and climate in its current balance. The greenhouse effect, or warming of the planet, is a consequence of an excess of atmospheric CO 2 caused by deforestation (reduced CO 2 consumption) and compounded by excessive fossil fuel energy use (increased CO 2 production). to Carbon is a critical element in the formation of stable humus. The carbon: nitrogen (C : N) ratio of the organic matter supplied to the soil is a controlling factor in this process. A ratio of about 20:1 is considered ideal. If greater amounts of carbon are present, decomposition slows as micro-organisms become nitrogen-starved and compete with the plants for available nitrogen. Nitrate nitrogen practically disappears from the soil because microbes need nitrogen to build their tissues. Carbon is present in reduced forms, such as methane (CH 4 ) and organic matter, and in more oxidized forms, such as carbon monoxide (CO) and carbon dioxide (CO 2 ). Electron donors (E.g., hydrogen, which is a strong reductant) and electron acceptors (E.g., O 2 ) influence the course of biological and chemical reactions involving carbon. Hydrogen can be produced when organic matter is degraded, especially under anoxic conditions when fermentation occurs. Although carbon cycles continuously from one form to another, for the sake of clarity, we shall say that the cycle begins with carbon fixation - the conversion of CO 2 into organic matter. Plants like trees and crops are often thought of as the principal CO 2 - fixing organisms, but at least half the carbon on Earth is fixed by microbes, particularly marine photosynthetic prokaryotes and protists (E.g., the cyanobacteria Prochlorococcus and Synechococcus, and diatoms, respectively). Carbon is also fixed by chemolithoautotrophic microbes. All fixed carbon enters a common pool of organic matter that can then be oxidized back to CO 2 through aerobic or anaerobic respiration and fermentation. 44 P a g e Compiled by: Derib A.

50 Alternatively, inorganic (CO 2 ) and organic carbon can be reduced anaerobically to methane (CH 4 ). Methane is produced by archaea in anoxic habitats. In a water column, the anoxic zone where methane is produced is often below anoxic zone. Therefore, as methane moves up the water column, it is oxidized before reaching the atmosphere. Archaea such as Methano brevibacter in the guts of termites contribute to methane production. Because microorganisms require each macronutrient, if an environment is enriched in one nutrient but relatively deficient in another, the nutrients may not be completely recycled into living biomass. For instance, chitin, protein, and nucleic acids contain nitrogen in large amounts. If these substrates are used for growth, the excess nitrogen and other minerals that are not used in the formation of new microbial biomass are released to the environment in the process of mineralization. This is the process by which organic matter is decomposed to release simpler, inorganic compounds (E.g., CO 2, NH + 4, CH 4, H 2 ). Most carbon substrates can be degraded easily with or without oxygen present, but this is not always the case. The exceptions are hydrocarbons and lignin. Hydrocarbons are unique in that microbial degradation most often involves the initial addition of molecular O 2. Anaerobic degradation of hydrocarbons proceeds more slowly and only in microbial communities that have been exposed to these compounds for extended periods. The carbon cycle has come under intense scrutiny in the last decade or so. This is because CO 2 levels in the atmosphere have risen from their preindustrial concentration of about 280 µmol per mol to 376 µmol per mol in This represents an increase of about one-third, and CO 2 levels continue to rise. Like CO 2, methane is also a greenhouse gas and its atmospheric concentration is likewise increasing about 1% per year, from 0.7 to 1.7 ppm (volume) since the early 1700s. These changes are clearly the result of the combustion of fossil fuels and altered land use. The term greenhouse gas describes the ability of these gasses to trap heat within Earth s atmosphere, leading to a documented increase in the planet s mean temperature. Indeed, over the past 100 years, Earth s average temperature has increased by 0.6 C and continues to rise at a rapid rate. 45 P a g e Compiled by: Derib A.

51 Fig. Carbon cycle: A good C:N ratio will result in the formation of both effective humus and stable humus. As decay occurs, the C:N ratio of the plant material decreases since carbon is being lost as CO 2, and nitrogen is conserved. This process continues until the micro-organisms run out of easily-oxidized carbon. The exuded, undecomposed carbon persists as stable humus. Fig. The Basic Carbon Cycle in the Environment. Carbon fixation can occur through the activities of photoautotrophic and chemoautotrophic microorganisms. Methane can be produced from inorganic substrates (CO 2 + H 2 ) or from organic matter. Carbon monoxide (CO)- produced by sources such as automobiles and industry- is returned to the carbon cycle by CO-oxidizing bacteria. Aerobic processes are noted with blue arrows, and anaerobic processes are shown with red arrows. 46 P a g e Compiled by: Derib A.

52 9.3. Organic Matter Decomposition and the Soil Food Web Soil organic matter When plant residues are returned to the soil, various organic compounds undergo decomposition. Decomposition is a biological process that includes the physical breakdown and biochemical transformation of complex organic molecules of dead material into simpler organic and inorganic molecules. Decomposition of organic matter is largely a biological process that occurs naturally. Its speed is determined by three major factors: soil organisms, the physical environment and the quality of the organic matter. In the decomposition process, different products are released: carbon dioxide (CO 2 ), energy, water, plant nutrients and resynthesized organic carbon compounds. Successive decomposition of dead material and modified organic matter results in the formation of a more complex organic matter called humus. This process is called humification. Humus affects soil properties. As it slowly decomposes, it colours the soil darker; increases soil aggregation and aggregate stability; increases the CEC (the ability to attract and retain nutrients); and contributes N, P and other nutrients. Soil organisms, including micro-organisms, use soil organic matter as food. As they break down the organic matter, any excess nutrients (N, P and S) are released into the soil in forms that plants can use. This release process is called mineralization. The waste products produced by micro-organisms are also soil organic matter. This waste material is less decomposable than the original plant and animal material, but it can be used by a large number of organisms Decomposition process Fresh residues consist of recently deceased micro-organisms, insects and earthworms, old plant roots, crop residues, and recently added manures. Crop residues contain mainly complex carbon compounds originating from cell walls (cellulo se, hemicellulose, etc.). Chains of carbon, with each carbon atom linked to other carbons, form the backbone of organic molecules. These carbon chains, with varying amounts of attached oxygen, H, N, P and S, are the basis for both simple sugars and amino acids and more complicated molecules of long carbon chains or rings. Depending on their chemical structure, decomposition is rapid (sugars, starches and proteins), slow (cellulose, fats, waxes and resins) or very slow (lignin). 47 P a g e Compiled by: Derib A.

53 Beneficial Property of Soil Organic Matter and Humus to Soil Improved fertilizer efficiency; Long life N for example, urea performs days longer; Improved nutrient uptake, particularly of P and Ca; Stimulation of beneficial soil life; Provides magnified nutrition for reduced disease, insect and frost impact; Salinity management - humates buffer plants from excess sodium; Organic humates are a catalyst for increasing soil C levels Nitrogen Cycle Like the carbon cycle, cycling of nitrogenous materials makes life on Earth possible. Nitrogen fixation is a uniquely prokaryotic process; apart from a limited amount of nitrogen fixation that occurs during lightning strikes, all organic nitrogen is of prokaryotic origin. Nitrogen fixation can be carried out under oxic and anoxic conditions. Microbes such as Azotobacter and the cyanobacterium fix nitrogen aerobically, while free-living anaerobes such as members of the genus Clostridium fix nitrogen anaerobically. Perhaps the best-studied nitrogen-fixing microbes are the bacterial symbionts of leguminous plants, including Rhizobium, its α-proteobacterial relatives, and some recently discovered β-proteobacteria (e.g., Burkholderia and Ralstonia spp.). However, other bacterial symbionts fix nitrogen. For instance, the actinomycete Frankia fixes nitrogen while colonizing many types of woody shrubs, and the heterocystous cyanobacterium Anabaenea fixes nitrogen when in association with the water fern Azolla. The product of N 2 fixation is ammonia (NH 3 ); it is immediately incorporated into organic matter as an amine. The addition of eight electrons per N atom requires a great deal of energy and reducing power. The nitrogenase enzyme is thus very sensitive to O 2 and must be protected from oxidizing conditions. Aerobic and microaerophilic nitrogen-fixing bacteria employ a number of strategies to protect their nitrogenase enzymes. For example, heterocystous cyanobacteria physically separate nitrogen fixation from oxygenic photosynthesis by confining the process to special cells called heterocysts, while other cyanobacteria fix nitrogen only at night when photosynthesis is impossible. N 2 + 8H e ATP 2NH 3 + H ADP + 16P i 48 P a g e Compiled by: Derib A.

54 Ammonia made by N 2 fixation is immediately incorporated into organic matter as amines. These amine N-atoms are eventually introduced into proteins, nucleic acids, and other biomolecules. The N cycle continues with the degradation of these molecules into ammonium (NH4 + ) within mixed assemblages of microbes. One important fate of this ammonium is its conversion to nitrate (NO - 3 ), a process called nitrification. This is a two step process whereby ammonium ion (NH4 + ) is first oxidized to nitrite (NO - 2 ), which is then oxidized to nitrate. The production of nitrate is important because it can be reduced and incorporated into organic nitrogen; this process is known as assimilatory nitrate reduction. The use of nitrate as a source of organic nitrogen is an example of assimilatory reduction. Because assimilatory reduction of nitrate to ammonium is energetically expensive, nitrate sometimes accumulates as a transient intermediate. Alternatively, for some microbes nitrate serves as a terminal electron acceptor during anaerobic respiration; this is a form of dissimilatory reduction. In this case, nitrate is removed from the ecosystem and returned to the atmosphere as dinitrogen gas (N 2 ) through a series of reactions that are collectively known as denitrification. This dissimilatory process, in which nitrate is used as an electron acceptor in anaerobic respiration, usually involves heterotrophs such as Pseudomonas denitrificans. The major products of denitrification include nitrogen gas (N 2 ) and nitrous oxide (N 2 O), although nitrite (NO - 2 ) also can accumulate. Nitrite is of environmental concern because it can contribute to the formation of carcinogenic nitrosamines. Finally, nitrate can be transformed to ammonia in dissimilatory reduction by a variety of bacteria, including Geobacter metallireducens, Desulfovibrio spp., and Clostridium spp. A recently identified form of nitrogen conversion is called the anammox reaction (anoxic ammonium oxidation). In this anaerobic reaction, chemolithotrophs use ammonium ion (NH + 4 ) as the electron donor and nitrite (NO - 2 ) as the terminal electron acceptor; it is reduced to nitrogen gas (N 2 ). In effect, the anammox reaction is a shortcut to N 2, proceeding directly from ammonium and nitrite without having to cycle first through nitrate. Although this reaction was known to be energetically possible, microbes capable of performing the anammox reaction were only recently documented. The discovery that marine bacteria perform the anammox reaction in the anoxic waters just below oxygenated regions in the open ocean solved a longstanding 49 P a g e Compiled by: Derib A.

55 mystery. For many years microbiologists wondered where the missing NH + 4 could be mass calculations did not agree with experimentally derived nitrogen measurements. The discovery that planctomycete bacteria oxidize measurable amounts of NH + 4 to N 2, thereby removing it from the marine ecosystem, has necessitated a reevaluation of nitrogen cycling in the open ocean. The N cycle is the most complex nutrient cycle. It occurs naturally in many chemical compounds, the simplest of which are nitrogen gas (N₂), ammonium (NH₄+), nitrite (NO₂ ), and nitrate (NO₃ ). The nitrogen present in living things, and in the matter produced by the decomposition of living things, is known as "organic nitrogen." The processes of the nitrogen cycle create these various molecules from each other. Nitrogen in its gaseous form (N₂) makes up the great majority (~78%) of the earth's atmosphere. Nitrogen is also essential to all life since it is present in all amino acids. But atmospheric nitrogen cannot be assimilated by most organisms. Natural fixation of the gaseous nitrogen is the process of conversion by which certain prokaryotes (bacteria) convert gaseous nitrogen into the forms that other organisms can use. Without Nitrogen, complex life could not exist on earth. The prokaryotes that fix atmospheric nitrogen as organic nitrogen often live in association with legumes (e.g., Rhizobium). But certain free-living prokaryotes can also carry out this conversion (for example, those in the genus Azotobacter). Denitrifying prokaryotes (shown at right in the diagram) complete the c ycle by converting organic nitrogen back into nitrogen gas. Although the N-cycle is very complex, it is probably the most important nutrient cycle to understand. There are two reasons for this: 1) N is usually the most growth-limiting plant nutrient in terrestrial (land) ecosystems, so there is often a very large crop-yield response to additional N, and 2) N in the nitrate form is very soluble and one of the most mobile plant nutrients in soil, so it can easily be lost from farm fields and become a contaminant in surface or groundwater. Managing N is a critical part of soil fertility management. 50 P a g e Compiled by: Derib A.

56 Fig. A Simplified Nitrogen Cycle. Flows that occur predominantly under oxic conditions are noted with open arrows. Anaerobic processes are noted with solid bold arrows. Processes occurring under both oxic and anoxic conditions are marked with cross-barred arrows. 51 P a g e Compiled by: Derib A.