Microbial Biotechnology Lec2 PDF

Microbial Biotechnology Dr. Eman Owis Lecturer Of Microbial Biotechnology – Mansoura Uni P h. D. G ö t t i n g e n U n i - G e r m a n y [email protected]  Microbial...

Microbial Biotechnology Dr. Eman Owis Lecturer Of Microbial Biotechnology – Mansoura Uni P h. D. G ö t t i n g e n U n i - G e r m a n y [email protected]  Microbial biotechnology Nanotechnology in Microorganisms Fermentation Genetically Modified Enzymes in different industry and Introduction associated with Biotechnology Products industry pharmatheutical industry industry & Bioethics Applications of Definition Bacteria, yeast and molds Introduction Genetic Engineering Introduction nanotechnology Production of medical Ethical aspects of Branches of Factors influencing and industrial medical and Fermenter DNA, RNA, Protein biotechnology microbial activity enzymes from pharmatheutical microorganisms biotechnology Importance of bacteria in Molecular Biology Different enzymes in History Types of fermentation the industry techniques medicine and industry Microbial Importance of Molds in biotechnology safety industry & Medicine and regulations Different techniques associated with Microbial biotechnology  The Scope of Microbiology Microbiology: The study of living things too small to be seen without magnification – Microorganisms or microbes- these microscopic organisms – Commonly called “germs, viruses, agents…” but not all cause disease and many more are useful or essential for human life  Introduction to Microbiology How Can Microbes Be Classified? – Carolus Linnaeus (Swedish) developed taxonomic system for naming plants and animals and grouping similar organisms together – Leeuwenhoek’s microorganisms grouped into six categories as follows: Fungi Protozoa Algae Bacteria Archaea Small animals  Fungi – Eukaryotic (have membrane-bound nucleus) – Obtain food from other organisms – Possess cell walls – Composed of Molds – multicellular; have hyphae; reproduce by sexual and asexual spores Yeasts – unicellular; reproduce asexually by budding; some produce sexual spores  Protozoa – Single-celled eukaryotes – Similar to animals in nutrient needs and cellular structure – Live freely in water; some live in animal hosts – Asexual (most) and sexual reproduction – Most are capable of locomotion by Pseudopodia – cell extensions that flow in direction of travel Cilia – numerous, short, hairlike protrusions that propel organisms through environment Flagella – extensions of a cell that are fewer, longer, and more whiplike than cilia  Algae – Unicellular or multicellular – Photosynthetic – Simple reproductive structures – Categorized on the basis of pigmentation, storage products, and composition of cell wall  Bacteria and Archaea – Unicellular and lack nuclei – Much smaller than eukaryotes – Found everywhere there is sufficient moisture; some found in extreme environments – Reproduce asexually – Two kinds Bacteria – cell walls contain peptidoglycan; some lack cell walls; most do not cause disease and some are beneficial Archaea – cell walls composed of polymers other than peptidoglycan  Viruses Not independently living cellular organisms Much simpler than cells- basically a small amount of DNA or RNA wrapped in protein and sometimes by a lipid membrane Individuals are called a virus particle or virion Depend on the infected cell’s machinery to multiply and disperse Factors influencing microbial activity Importance of Microbes in Biotechnology  Food Biotechnology Main Branches Water & Sewage Biotechnology Biodegradation and sewage treatment Environmental Biotechnology Nitrogen Fixation and Bio-Pesticide Industrial Biotechnology Production of valuable products Water & Sewage Biotechnology Biodegradation "Transformation of a substance into new compounds through biochemical reactions or the actions of microorganisms such as bacteria.“ Microbial Biodegradation is the use of bioremediation and biotransformation methods to utilize the naturally occurring ability of microbial xenobiotic metabolism to degrade environmental pollutants and transform or accumulate the environmental pollutants. Hydrocarbons (e.g. oil) Polychlorinated biphenyls (PCBs) Polyaromatic hydrocarbons (PAHs) Heterocyclic compounds (such as pyridine or quinoline) Pharmaceutical substances. Oil biodegradation Petroleum oil contains aromatic compounds that are toxic to most life forms. Episodic ‫ عرضيه‬and chronic ‫ مزمن‬pollution of the environment by oil causes major disruption to the local ecological environment. Marine environments in particular are especially vulnerable, as oil spills near coastal regions and in the open sea are difficult to be controlled. In addition to pollution through human activities, approximately 250 million liters of petroleum enter the marine environment every year from natural leaking. Alcanivorax borkumensis was the first obligate hydrocarbonoclastic bacteria (OHCB) bacteria to have its genome sequenced. In addition to hydrocarbons, crude oil often contains various heterocyclic compounds, such as pyridine, which appear to be degraded by similar mechanisms to hydrocarbons. Environmental Biotechnology 1- Nitrogen Fixation Nitrogen fixation is a process by which nitrogen (N2) in the atmosphere is converted into ammonia (NH3). Atmospheric nitrogen or molecular nitrogen (N2) is relatively inert: it does not easily react with other chemicals to form new compounds. The fixation process free up the nitrogen atoms from their di-atomic form (N2) to be used in other ways. Nitrogen fixation, natural and synthetic, is essential for all forms of life because nitrogen is required to biosynthesize basic building blocks of plants, animals, and other life forms, e.g., nucleotides for DNA and RNA and amino acids for proteins.  Two kinds of nitrogen fixers are recognized: free-living (non-symbiotic) bacteria, as Azotobacter, and mutualistic (symbiotic) bacteria such as Rhizobium, associated with leguminous plants. N2 Fixation in microalgae Environmental Biotechnology 2- Bio-pesticide Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals. Microbial pesticides consist of a microorganism (e.g., a bacterium, fungus, virus or protozoan) as the active ingredient. Microbial pesticides can control many different kinds of pests, although each separate active ingredient is relatively specific for its target pest[s]. The most widely used microbial pesticides are subspecies and strains of Bacillus thuringiensis, or Bt. Each strain of this bacterium produces a different mix of proteins and specifically kills one or a few related species of insect larvae.  Some Bt ingredients control moth larvae found on plants, other Bt ingredients are specific for larvae of flies and mosquitoes. The target insect species are determined by whether the particular Bt produces a protein that can bind to a larval gut receptor, causing the insect larvae to starve. How does Bacillus thuringiensis (Bt) work? Bt makes toxins that target insect larvae when eaten. In their gut, the toxins are activated. The activated toxin breaks down in their gut, and the insects die of infection and starvation. Death can occur within a few hours or weeks. The different types of Bt create toxins that can only be activated by the target insect larvae. What are some products that contain (Bt)? Currently, Bt strains are found in over 180 registered pesticide products. Bt products are used on crops and ornamental plants. Others are used in and around buildings, in aquatic settings, and in aerial applications. These products are commonly sprays, dusts, granules, and pellets. Some of these products are approved for use in organic agriculture. Some crops have been engineered to make the Bt toxin. These plant- incorporated protectants include corn, cotton, and soybeans. What happens to (Bt) when it enters the human body? When eaten, Bt is confined to the gut. It does not reproduce, and the toxin is broken down like other proteins in the diet. Bt leaves the body within 2 to 3 days. If breathed in, Bt can move to the lungs, blood, lymph, and kidneys. Bt is then attacked by the immune system. Levels of Bt decrease quickly one day after exposure. What happens to (Bt) in the environment? Toxins created by Bt are rapidly broken down by sunlight and in acidic soil. Other microbes in soil can also break it down. Bt does not readily leach in soil. It typically remains in the top several inches of soil. Industrial Biotechnology 1- Microbial Based Perfumes The flavor and fragrance industry experienced a shortage of patchouli oil in 2010 when soggy weather gave Indonesian growers a poor harvest of Pogostemon cablin, a perennial shrub in the mint family that is the source of the fragrant oil. That disappointment was followed by volcanic eruptions in the islands, which spawned earthquakes and a tsunami, further disrupting supply.  It is no wonder, then, that purchasers of fine-smelling and –tasting substances would seek alternatives to nature-grown materials. Indeed, major flavor and fragrance houses such as Givaudan are intrigued by the possibility of using biotechnology to produce key components of essential oils from abundant sugar feedstock via fermentation. So, the microbial platforms can produce just about any plant-derived molecule. Once they scale up, they say, supply shortages will be a thing of the past. Some example for scent and flavor by microorganisms: a-Diacetyl Diacetyl (CH3COCOCH3) "strong typical ‘butter’ odor and flavor on dilution" produced from acetoin by microbiological oxidation. (Bacteria: Lactococcus lactis, Lactobacillus sp., Streptococcus thermophilus, and Leuconostoc mesenteroides). A method for increasing the diacetyl production from bacteria such as S. diactilactis, S. cremoris and S. lactis has been patented. The use of humectants such as glycerol or sucrose lowers the water activity of the medium and results in greater diacetyl production.  The production of diacetyl is further enhanced by a low pH (less than 5.5), low temperature, and aeration. B- Vanillin Vanillin is a unique flavor chemical that occurs in Vanilla planifolia beans. Although vanillin can be chemically synthesized, but there is an increasing demand for natural vanillin. The direct extraction of vanillin from vanilla beans is expensive and limited which makes this compound a promising target for biotechnological flavor production.  Vanilla flowers are greenish-yellow, with a diameter of 5 cm (2 in). They last only a day and must be pollinated manually, during the morning, if the fruit is desired. Pollination simply requires a transfer of the pollen from the another to the stigma. If pollination does not occur, the flower is dropped the next day. In the wild, there is less than a 1% chance that the flowers will be pollinated, so to receive a steady flow of fruit, the flowers must be hand- pollinated when grown on farms.  Vanillin is also produced as an intermediate compound in the microbial degradation of several substrates such as ferulic acid, phenolic stilbenes, lignin, eugenol, and isoeugenol. Several bacterial and fungal strains of Pseudomonas putida, Aspergillus niger, Corynebacterium glutamicum, Corynebacterium sp., Arthrobacter globiformis, and Serratia marcescens are capable of conversion of natural eugenol and isoeugenol from essential oils into vanillin. Eugenol vanillin C- Antibiotics Antibiotics are antimicrobial agents produced naturally by other microbes. The first antibiotic was discovered in 1896 from the filamentous fungus Penicilium notatum. Penicillin was the first important commercial product produced by an aerobic, submerged fermentation e.g. penicillin, ampicillin, amoxicillin, …. It can Inhibit enzymes involved in the synthesis of peptidoglycan for bacterial cell walls, causing cell lysis or can be Bactericidal. Other antibiotics may affect: Cell membrane, DNA replication, Transcription, Translation Antibiotic production There are over 10,000 different antibiotics known, but only about 200 in commercial use, since most new antibiotics are no better than existing ones. There is a constant search for new antibiotics. Antibiotics are the most- prescribed drugs and are big business. Finding a new antibiotic and getting it on to the market is a very long process and can take 15 years. Antibiotic Production Methods Antibiotics are produced on an industrial scale using a variety of fungi and bacteria. Penicillin is produced by the fungus Penicillium chrysogenum which requires lactose, other sugars, and a source of nitrogen (in this case a yeast extract) in the medium to grow well. Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase. It requires a batch fermenter, and a fed-batch process is normally used to prolong the stationary period and so increase production.  Downstream processing is relatively easy since penicillin is secreted into the medium (to kill other cells), so there is no need to break open the fungal cells. However, the product needs to be very pure, since it being used as a therapeutic medical drug, so it is dissolved and then precipitated as a potassium salt to separate it from other substances in the medium. The resulting penicillin (called penicillin G) can be chemically and enzymatically modified to make a variety of penicillins with slightly different properties. These semi-synthetic penicillins include penicillin V, penicillin O, ampicillin and amoxicillin. Antibiotic production: 1.What is the Carbon source? 2.What is the nitrogen source? 3.What is the energy source? 4.Is the fermentation aerobic or anaerobic? 5.What is the optimum temperature? 6.Is penicillin a primary or secondary metabolite? 7.When is penicillin produced? 8.What was the first fungus known to produce penicillin? 9.What species produces about 60mg/dm3 of penicillin? 10.How did scientists improve the yield still further? 11. Why is batch culture used? 12. What are the processes involved in downstream processing? a) b) c) 13. How does Penicillin kill bacteria? 14. Why are Gram-negative bacteria not killed by penicillin? Any Questions