Microbial Biotechnology PPT 44 WDO2 (1) PDF

Addis Ababa Science and Technology University Department of Biotechnology COURSE TITLE: MICROBIAL BIOTECHNOLOGY COURSE NO. - Bot3105 CREDIT HRS-3 Million Yohannes Senior Lecturer  INTRODUCTION TO MICROBIAL BIOTECHNOLO...

Addis Ababa Science and Technology University Department of Biotechnology COURSE TITLE: MICROBIAL BIOTECHNOLOGY COURSE NO. - Bot3105 CREDIT HRS-3 Million Yohannes Senior Lecturer  INTRODUCTION TO MICROBIAL BIOTECHNOLOGY Microbial Biotechnology can be defined as a technology that uses microorganisms or derivatives to make products of industrial and medical applications. The microbial biotechnology can be simply referred as a fermentation process in which natural substrates of microbes are converted into value added products. The microorganisms exploited are natural, laboratory selected mutant or genetically engineered strains. In general, microorganisms are capable of producing an array of valuable compounds ranging from macromolecules such as proteins, nucleic acids and carbohydrates to smaller molecules called metabolites in small amounts. These metabolites produced by such microorganisms are further classified as primary and secondary metabolites.  INTRODUCTION TO MICROBIAL…cont The primary metabolites are produced by the microbes during their energy metabolism and are essential for the vegetative growth, development and reproduction of the organisms. It includes carbohydrates, vitamins, proteins and amino acids. The secondary metabolites are produced after the growth of the microbes and usually during the stationary phase of the growth of the microorganism. It includes antibiotics, alkaloids and toxins. The microbial population produces such compounds for their own benefits, but those are exploited scientifically to produce products of potential application in human therapeutics and also for many other uses. This could be made possible through genetic engineering techniques or rDNA technology.  INTRODUCTION TO MICROBIAL…cont The insulin production by rDNA technology. The microbial products such as enzymes, vitamins, alcohols, amino acids, recombinant proteins, fertilizers and biopesticides are of economically valuable with good market value. For industrial production, the micro organisms are selected for their metabolic activities to produce one or more specific products in high rate. The microbes are also utilized in areas such as waste water treatment, Microbial Enhanced Oil Recovery (MEOR), biodegradation and biomining. With these aspects microbial biotechnology play an exemplary role in our lives and life spans. 1.1 Brief on Microbial physiology and ecology Microbial physiology Physiology describes the functioning of unicellular microorganisms in terms of correlations between systems properties. In microbiology, microbial physiology would determine how the flux of catabolism of a given substrate varies with the change in environmental conditions such as pH, water activity, temperature, etc  1. Cell Structure Prokaryotic vs. Eukaryotic Cells: Prokaryotes (e.g., bacteria) lack membrane-bound organelles, while eukaryotes (e.g., fungi, protozoa) have them. Cell Wall Composition: Bacteria have peptidoglycan in their cell walls; archaea have pseudopeptidoglycan or other polymers. The interface between the microbial cell and its external environment is the cell surface. It protects the cell interior from external hazards and maintains the integrity of the cell  Cell Wall. The surface of gram-negative cells is much more complex than that of gram-positive cells. The gram- positive cell surface has two major structures: the cell wall and the cell membrane. The cell wall of gram-positive cells is composed of multiple layers of peptidoglycan, which is a linear polymer of alternating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).  2. Metabolism Energy Sources: Microbes can be classified based on their energy sources: – Phototrophs: Use light (e.g., cyanobacteria). – Chemotrophs: Obtain energy from chemical compounds (e.g., sulfur bacteria). (Organothrophs vs lithotrophs) Carbon Sources: – Autotrophs: Use CO₂ (e.g., some bacteria). – Heterotrophs: Require organic compounds (e.g., most bacteria and fungi).  Metabolism The cell can generate ATP in two ways: (1) by substrate-level phosphorylation and oxidative phosphorylation Respiration and Fermentation Aerobic Respiration: Requires oxygen and produces ATP via the electron transport chain.  Metabolism Anaerobic Respiration: Occurs without oxygen, using alternative electron acceptors (e.g., nitrate, sulfate). Fermentation: An anaerobic process that converts sugars to acids or alcohol; used by yeast and some bacteria. Table: Variation in fermentation products formed from Pyruvate  Endospores Endospores provide resistance to heat, desiccation, radiation, and other environmental factors that may threaten the existence of the organism. Endospores also provide a selective advantage for survival and dissemination of the species that produce them.  Bacterial Growth Mastering the fundamentals of bacterial growth allows the researcher to change culture conditions artificially, adjust the bacterial phases of growth and reproduction, and use beneficial bacteria more efficiently. Bacterial growth involves inoculating a certain number of bacteria into a suitable liquid medium and checking the number of viable cells at different time intervals. With the collected information, it is possible to generate a growth curve using culture time as the horizontal axis and the logarithmic number of viable cells in the culture as the vertical axis. Growth curves can generally be divided into four major phases. Binary Fission Reproduction  Stress Responses Osmotic Stress: Temperature Stress Table showing pH ranges of Bacterial growth.  Genetic Regulation Gene Expression: Controlled by factors like promoters and repressors; operons Horizontal Gene Transfer: transformation, transduction, and conjugation allow microbes to share genes, antibiotic resistance. Biofilms Formation: Microbes adhere to surfaces and each other, forming complex communities. Advantages: Enhanced protection Signaling--Quorum Sensing  Microbial ecology Microbial ecology is the study of the relationships between microorganisms and their environment, including interactions Microbes play crucial roles in nutrient cycling Basic Definition in the Study of Microbial Ecology Ecosystem: This is a combination of biotic and abiotic components Community This is a collection of microorganism inhabiting a given site in the ecosystem ( Population: Individual:  Habitat area having a degree of uniformity in terms of the abiotic components. Niche. The ability of microorganisms to make use of resources available in their habitat.Those with narrow niche are highly specialized and perform single function or role e.g. obligate parasites, autotrophic organisms, while those that carryout a range of functions are said to have a broad niche e.g. heterotrophic organism.  Habitat Those that occupy a narrow niche are easily eliminated if there is a change in environmental conditions especially as it affects their survival  COLONIZATION AND SUCCESSION The first microorganisms to arrive on such surfaces are called pioneers. These organisms grow and multiply to form the pioneer community and from the time the exposed areas are occupied by microorganisms, it is said to be colonized.  COLONIZATION AND SUCCESSION the pioneer community, they feed on the substrates, produced byproducts and other waste materials or metabolites and so the environment becomes modified. The modified environment paves way for colonization by other organisms, while the pioneer communities are gradually eliminated. This phenomenon is referred to as succession.  Climax Community. When colonization has taken place, succession follows and there is a continuous modification of the environment. However, at a stage the community remains constant for at least some time and the process of succession is stopped; and a community that characteristics that habitat results. This is called the climax community the microorganisms and the physical environment are in constant equilibrium  Climax Community Often, there are alternations in the physical conditions e.g when a large quantity of pollutants is introduced into a stream, the organic matter component is eliminated and the climax community is distorted. However, such disturbances are temporary and the original climax community is restored with time or as soon as the disturbance is removed.  I. Types of Microbial interactions with Biotic component Microbial interactions with biotic components can be categorized into several types, each with distinct characteristics and outcomes. a. Mutualism Both organisms benefit from the interaction. Examples: – Mycorrhizal Fungi and Plants: Fungi enhance nutrient uptake (especially phosphorus) for plants, while plants provide carbohydrates to fungi. – Rhizobia and Legumes: Rhizobia bacteria fix atmospheric nitrogen for legumes, benefiting from carbohydrates produced by the plants. b. Commensalism One organism benefits while the other is neither helped nor harmed. Examples: – Skin Microbiota: Bacteria living on human skin obtain nutrients from sweat and oils without affecting the host. – Birds and Tree Sap: Birds may feed on tree sap without causing harm to the tree.  Types of Microbial interactions c. Parasitism One organism benefits at the expense of the other. Examples: – Pathogenic Bacteria: Bacteria like Staphylococcus aureus can infect humans, causing disease while deriving nutrients from the host. – Fungi and Insects: Entomopathogenic fungi infect and kill insects, using them as a nutrient source. 2. Competitive Interactions Microorganisms compete for limited resources such as nutrients, space, or light. Examples: – Bacterial Competition: Different bacterial species in soil compete for nitrogen or carbon sources by producing antibiotics against competitors, influencing community structure. Algal Blooms: In aquatic environments, certain algae can out-compete others for light and nutrients, leading to dominance in a given area  Types of Microbial interactions 3. Predation Microorganisms can prey on other microorganisms, influencing population dynamics. Examples: – Bacteriophages: Viruses that infect and lyse bacteria, regulating bacterial populations. – Protozoan Predators: Protozoa such as Amoeba consume bacteria, affecting microbial community structure. 4. Competition vs. Cooperation Balance: Microbial communities often exhibit a balance between competitive and cooperative interactions, which can shift based on environmental conditions. Community Dynamics: Changes in resource availability can lead to shifts from competitive to cooperative interactions.  Types of Microbial interactions 5. Co-infection and Synergy Multiple microbial species infecting the same host can interact synergistically or antagonistically. Examples: – Polymicrobial Infections: Infections involving multiple bacterial species (e.g., in chronic wounds) can be more severe than infections with a single species. – Synergistic Infections: Certain bacteria may enhance the virulence of others, as seen in some cases of respiratory infections. 6. Biofilms and Community Behavior Microbes can form biofilms, which are structured communities of microorganisms adhering to surfaces. Interactions: – Cooperation: In biofilms, different species can cooperate for nutrient acquisition and protection. – Communication: Quorum sensing allows microbes to communicate and coordinate behavior within biofilms.  Types of Microbial interactions 7. Impact on Host Health Beneficial Microbiota: Microbial communities in the human gut play crucial roles in digestion, immunity, and overall health. Pathogenic Microbes: Pathogens can disrupt normal microbial communities, leading to dysbiosis and disease. 8. Interspecies Signaling Chemical Communication: Microorganisms can produce signaling molecules to communicate with one another, influencing behavior and interactions. Examples: – Quorum Sensing: Bacteria can sense their population density and coordinate group behaviors, such as biofilm formation or virulence factor production.  II. Microbial Adaptations to Abiotic Factors Microbes have evolved various adaptations to thrive in diverse abiotic conditions: a. Temperature Psychrophiles: Thrive in cold environments (0-20°C). Mesophiles: Optimal growth at moderate temperatures (20-45°C). Thermophiles: Prefer high temperatures (45-80°C), often found in hot springs. b. pH Levels Acidophiles: Grow optimally in acidic conditions (pH < 5). Alkaliphiles: Thrive in alkaline environments (pH > 9). Neutralophiles: Favor neutral pH (around 7), common in most environments. c. Moisture Hygrophiles: Require high moisture levels to grow. Xerophiles: Adapted to dry environments, can survive with minimal water.  Microbial Adaptations 3. Nutrient Availability Nutrient Cycling: Microbes play vital roles in recycling nutrients, such as carbon, nitrogen, and phosphorus, through processes like decomposition. Nutrient Limitation: Microbial growth can be limited by the availability of essential nutrients (e.g., nitrogen or phosphorus), affecting ecosystem productivity. 4. Microbial Interactions with Soil Soil Microbiome: A complex community of microbes that interact with soil minerals, organic matter, and plants. Soil Structure: Microbial activity affects soil structure and stability, influencing water retention and aeration.  Microbial Interactions with Soil.. The soil (as an ecosystem) is the natural medium for terrestrial plant growth. It is composed of varying proportions of organic and inorganic components which arise …weathering of rocks, decomposition of plants and animals materials, and redistribution of materials by water movement and human activities. Like many other types of environments, microorganisms (bacteria, fungi, algae, nematodes, actinomycetes, and protozoa) are also present in the soil and they are known to perform various functions and even contribute to the structure of the soil by producing gums and cement which apart from holding the soil together can also damage the structures by blocking the pores.  Microbial Interactions with Soil Fungi are abundant in the soil, however not as bacteria. Different soils have different types of fungal species associated with it depending on the prevailing conditions. Fungi are active in the soil as mycelia and are usually dormant as spores. One of the most important functions of soil fungi is the degradation of complex plant structures like hemicelluloses, pectin, lignin, cellulose etc into simple molecules which are made available to plants as nutrients. They help in the formation of stable soil by binding the soil through hyphal penetration. Fungi are able to breakdown complex proteinous materials, producing ammonia and sulphur compounds which could be used by higher plants. Fungal degradation activities improves soil texture and organic composition Mycorrhizal symbiotic fungi provide different minerals to plant roots by absorbing minerals from the soil Soil fungi may function as parasites in the soil under certain conditions Often, fungi compete with higher plants for nutrients. Under certain conditions, they are able to trap nematodes and protozoa (biological control).  Microbial Interactions with Soil Bacteria are known to be the most abundant type of soil micro-organisms. Hundreds of species of bacteria are present in 1gram of soil and they vary in different soils found in various parts of the world. The nature and extent of soil bacterial population however depends on prevailing environmental conditions, such as moisture, pH, temperature, aeration and nutrient availability. Soil fertility is largely dependent on bacterial activity, and soil bacteria are essential to all life processes because without putrification and decay, there will be no decomposition of dead plants and animal matter.  Microbial Interactions 5. Microbial Interactions with Water Aquatic Ecosystems: Microbes are integral to nutrient cycling in freshwater and marine environments, influencing water quality and ecosystem health (Natural purification). Hydrodynamics: Water flow and turbulence affect microbial distribution and biofilm formation. 6. Microbial Influence on Biogeochemical Cycles Carbon Cycle: Microbes decompose organic matter, releasing carbon dioxide back into the atmosphere or converting it into biomass. Nitrogen Cycle: Microbes are involved in nitrogen fixation, nitrification, denitrification, and ammonification, regulating nitrogen availability in ecosystems. Sulfur Cycle: Sulfate-reducing and sulfur-oxidizing bacteria play key roles in sulfur transformations. 7. Microbial Interactions with Minerals Mineral Weathering: Microbes contribute to the weathering of rocks, releasing minerals essential for plant nutrition. Biomineralization: Some microbes can precipitate minerals, influencing soil and sediment composition.  Microbial Interactions 8. Impact of Environmental Stressors Pollution: Heavy metals and chemicals can affect microbial communities, leading to shifts in diversity and function. E.g. Cadmium (Cd), Chromium (Cr), Iron (Fe), Manganese (Mn) and Zinc (Zn), Pb Climate Change: Changes in temperature and precipitation patterns can alter microbial distribution and interactions with abiotic factors. 9. Biofilm Formation on Abiotic Surfaces Adhesion: Microbes can attach to surfaces (e.g., rocks, pipes) and form biofilms, which can alter the physical and chemical properties of their environment. Nutrient Capture: Biofilms can enhance nutrient uptake from the surrounding environment.  1.2 The use and application of microbes in biotechnology Microbes are used to synthesize a wide range of products that are highly valuable to human beings. Such microbes are industrially exploited to produce products such as food additives, beverages and therapeutics for human and animal health. The industrially exploited microorganisms are called as industrial microorganisms. It mostly includes bacteria and fungi. 1. Medical Applications Pharmaceutical Production: – Antibiotics: Produced by fungi (Penicillium) and bacteria (Streptomyces). – Health: Insights into human microbiome interactions can inform treatments for various diseases and promote health. – Vaccines: Recombinant DNA technology allows for the production of vaccines (e.g., hepatitis B vaccine using yeast). Diagnostics: – Microbes used in tests to detect diseases (e.g., bacterial culture for infections).  The use and application.. 2. Environmental applications Bioremediation: Utilizing microbes to degrade pollutants in soil and water(e.g., oil spills, heavy metals)in contaminated environments, often influenced by abiotic factors like soil composition and moisture. /Case studies/ Waste Treatment: Microbial processes in wastewater treatment plants to break down organic matter and remove contaminants. 3. Agricultural applications. Agriculture: Understanding microbial interactions with soil and nutrients can enhance crop yields and sustainability.  The use and application.. 4. Industrial applications Microorganisms are used for synthesis of different chemicals which are beneficial to human. Biofuels: – Ethanol production from fermentation of sugars by yeasts (Saccharomyces cerevisiae). – Microbial production of biodiesel from fatty acids. Enzyme Production: – Microbes produce enzymes for various industrial applications, such as amylases in baking and proteases in detergents.  Table Microorganisms employed for Production of Industrial Products Products Microorganisms Employed Microbial Product Antibiotics Streptomyces griseus, Streptomyces parvullus StreptomycinActinomycin D Brevibacterium flavum Cornyebacterium Lysine Glutamic Acid Amino Acids glutamicum Beverages Saccharomyces cerevisia, Saccharomyces uvarum Wine Beer Organic Lactobacillus sp. Gluconobacter suboxidans Lactic Acid Gluconic Acids Acid Vitamins lakeslea trispora Ashbya gossypii Lysine Glutamic Acid Organic Saccharomyces cerevisiae Clostridium Ethanol Acetone Solvents acetobutylicum Food Penicillium roquefortii Lactobacillus bulgaricus Cheese Yogurt Products Enzymes Aspergillus oryzae Aspergillus niger Amylase Cellulase Pharmaceutic Trichoderma polysporum Cyclosporin A al Drug  The use and application.. 5. Agricultural Biotechnology Biopesticides: – Use of beneficial microbes (e.g., Bacillus thuringiensis) to control pests naturally. Biofertilizers: – Nitrogen-fixing bacteria (e.g., Rhizobium) enhance soil fertility. – Phosphate-solubilizing microbes make phosphorus available to plants. Plant Growth Promotion: – Plant Growth Promoting Rhizobacteria (PGPR) enhance plant growth and health. 6. Food Biotechnology Fermentation: – Microbes used in the production of yogurt, cheese, bread, and fermented vegetables. Probiotics: – Beneficial live microorganisms that improve gut health, found in fermented foods.  The use and application.. 7. Synthetic Biology Genetic Engineering: – Modification of microbial genomes for desired traits, such as enhanced production of pharmaceuticals. Metabolic Engineering: – Reprogramming metabolic pathways to increase yields of valuable compounds (e.g., bio-based materials). 8. Future Perspectives Innovative Applications: – Research into personalized medicine and sustainable agricultural practices using microbes. Advancements in Technology: – Improvements in genomics and bioinformatics enhance understanding of microbial functions and their applications.  1.3 Scopes of Microbial Biotechnology 1.3.1 In Human Therapeutics The major application of microbial biotechnology in human therapeutics. Production of recombinant protein and antibiotics for therapeutics. – Insulin, the first genetically engineered therapeutic agent is used to treat diabetes. It is expressed from human insulin genes on plasmid vector inserted into E. coli. – Production of human growth hormone (hgH) for dwarf individuals is also an important application. The human growth hormone is secreted in the pituitary gland of humans. Reduction in the hormone level causes dwarfism. Genetically engineered hgH is cost effective and safe to use. – Human tissue plasminogen activator is another therapeutic agent. It is proteolytic enzyme that cleaves single peptide bond in plasminogen to form plasmin. This plasmin has the capacity to degrade fibrin clots and hence it is widely used in the treatment of acute myocardial infarction.  Scopes of Microbial Biotechnology – Production of DNA vaccines is another application of microbial biotechnology. It induces both humoral and cellular responses. – The secondary metabolites produced by the microorganisms are utilized for the production of various antibacterial and anticancer drugs. – Microorganism, Streptomyces avermitilis is used in the production of series of drug called Avermectin which is used to treat parasitic worms. – Microorganisms are also industrially used for the production of vaccines, antibiotics and diagnostic kits. The Streptococcus sp. produces an enzyme called Streptokinase which acts as clot buster for removing blood clots.  Scopes of Microbial … 1.3.2 In Agriculture Microbial biotechnology plays an inevitable role in the field of agriculture. The microorganisms are widely exploited for the production of Biofertilizers and microbial pesticides, insecticides and herbicides. Biofertilizers are substances produced from microorganisms such as bacteria, fungi and algae. These microorganisms help the plants to absorb nutrients and fix atmospheric nitrogen into soil. Biofertilizers are cost effective, environmentally safe and increase crop production. Hence it is recommended over chemical fertilizers. – Bacterial Biofertilizers Eg: Rhizobium, Azospirillium and Azotobacter. – Fungal Biofertilizers Eg: Mycorrhiza – Algal Biofertilizers Eg: Azolla  Scopes of Microbial… Microbial pesticides are composed of naturally occurring bacteria, virus and fungi and are used to control and kill pests that affect the plant growth. Generally prolonged use of chemical pesticides affects the soil fertility and imposes adverse effects on human beings and animals. In an alternative view, this microbial pesticide promotes soil fertility and is ecologically safe since it is produced from microorganisms. Microbial insecticides are also composed of naturally occurring microorganisms and are used to control insect pests. It is a part of Integrated Pest Management (IPM). Several species of Bacillus bacteria are commonly utilized for the production of insecticides. Bacillus thuringiensis, a Gram negative bacterium is widely used to kill insects and other arthropods. Virus like Baculovirus is utilized as a potential candidate for insecticide production. Entomopathogenic fungi is also employed for insecticide production which are host specific and in the process other beneficial insects do not get affected.  Scopes of Microbial… Microbial herbicides are used to control the weeds that hinder the growth of the crop. The weeds affect the crop productivity and causes economical loss. In this approach, pathogens are isolated from weeds and are cultured to form infective propagules. These infective propagules when applied on the field targets and suppresses the growth of the target weeds.  Scopes of Microbial… 1.3.3 In Food Technology Microorganisms are used for the production of fermented foods and beverages. The fermenting property of the microorganisms makes it an ideal one for the production of food products such as cheese, bread and yoghurt. Fermented Foods Microorganism employed for Production Bread Saccharomyces cerevisiae Cheese Penicillium roquefortii Yoghur Lactobacillus bulgaricus Soy Sauce Aspergillus soyae Cakes Saccharomyces cerevisiae Table 1.1 Fermented Food Products and Microorganisms employed for their Production  Scopes of Microbial… The microorganisms used for the production influence of the nature of the food like flavour and odour. The nutritive content of the fermented food is improved by the presence of microorganisms. Hence the fermented foods are highly nutritive and easily digestible.  Scopes of Microbial… 1.3.4 In SCP Single Cell Protein refers to the total protein extracted from microbial cell cultures. It serves as nutritional supplements for both humans and animals. It is rich in protein content but it also contains carbohydrates, vitamins and minerals. Hence it is considered as nutritionally rich supplement. Several microorganisms like bacteria, yeast, fungi and algae are utilized for the production of single cell protein. Substrates like molasses, whey, cellulose, animal manures are used for the production of SCP. The microorganism utilizes these substrates as nutrients required for their growth and produce increased biomass concentrations. Microorganisms utilized for SCP production are as follows Bacteria: Pseudomonas fluorescens Algae: Spirulina sp. Fungi: Aspergillus niger, Aspergillus fumigates, Yeast: Saccharomyces cerevisiae, Candida utilis  Applications of SCP It is used as supplemented food for undernourished children. /stunted child../ It prevents accumulation of cholesterol in the body. It lowers blood sugar levels in diabetic patients  Scopes of Microbial… 1.3.5 Bioremediation It is the process of removal of pollutants from the environment through the application of microorganisms. The microorganisms utilized for the process are generally thermophilic anaerobic microorganisms and are used to remove organic wastes from the environment. The microorganisms degrade the environmental contaminants into less toxic forms through reactions that take place as a part of their metabolic process. Other interesting fact is that the microorganisms also possess some enzymes that allow the organism to utilize the contaminants as food for their growth. The effectiveness of the process depends on the environmental condition that allows microbial growth and activity.  Scopes of Microbial… 1.3.6 Biomining It is the process of cleaning up the sites that are polluted with metals. It can also be referred as the process of extracting economically valuable metals from rock ores and mine wastes. There are two methods employed in Biomining such as Bioleaching and Bio- oxidation. Bioleaching refers to the removal of metals from low grade ores and mineral concentrates. Usually the metals are found binded with the surface of the solid mineral and the microbes are able to oxidize that metal and make it dissolve in water. As the metals are dissolved in water, this method is called Bioleaching. This process is usually done with the application of microorganisms that are single celled and undergo chemosynthetic metabolism, for example mesophiles, the organisms that grows in moderate temperature and extremophiles, the organisms that grows in high pressure and temperature. Copper, zinc, cobalt and uranium are the metals extracted by this process. On the other hand, some metals are not dissolved by the microbes. In such case, the minerals surrounding the metals are decomposed by the microbes allowing the recovery of metal directly from the site. This process also involves oxidation but difference here is that the metal of interest is solubilized.  Scopes of Microbial… 1.3.7 Waste Water Treatment Waste water treatment technology is also an application in which toxic materials such as textile dyes, heavy metals and pesticides are removed by the microorganisms. Diagnostics are developed to detect disease causing organisms in the water so as to ensure the water quality. 1.3.8 Biosensors The genetically engineered microorganisms are exploited as living sensor to detect toxic chemicals in the soil, air and biological specimens. Toluene, a toxinogenic chemical found in chemical and radiation waste sites are also degraded by the application of microbial population.  Chapter 2: Important microorganisms for biotechnological applications 2.1 Criteria for selecting microbes for applications Microorganisms are selected for specific industrial applications based on their metabolic capabilities and growth conditions. Microorganisms, such as bacteria, fungi, and yeasts, play a crucial role in various industrial applications, including food production, pharmaceuticals, waste treatment, and bio-fuel production. The selection of these microorganisms is a meticulous process that depends on their metabolic capabilities and the conditions under which they can grow and thrive. The metabolic capabilities of a microorganism refer to its ability to convert raw materials into desired products. For instance, certain strains of yeast are used in the brewing industry because of their ability to ferment sugars into alcohol. Similarly, specific bacteria are used in the production of antibiotics due to their ability to produce these compounds as part of their metabolic processes. The metabolic capabilities of microorganisms are often determined through laboratory testing and genetic analysis.  2.1 Criteria for selecting microbes The growth conditions of a microorganism are also a key factor in their selection for industrial use. These conditions include factors such as temperature, pH, and the presence of specific nutrients or inhibitors. For example, thermophilic bacteria, which can survive at high temperatures, are used in industrial processes that require heat, such as certain types of waste treatment. Similarly, microorganisms that can survive in acidic or alkaline conditions may be used in industries where these conditions are prevalent. In addition to these factors, the selection of microorganisms for industrial applications may also consider their ease of cultivation and manipulation. Some microorganisms can be easily grown in large quantities, making them ideal for industrial use. Others can be genetically modified to enhance their metabolic capabilities or to make them more suitable for specific industrial processes.  2.1 Criteria for selecting microbes Furthermore, the safety of the microorganism is also a crucial consideration. Microorganisms used in food production or pharmaceuticals, for example, must be non-pathogenic and not produce harmful by-products. Regulatory bodies often have strict guidelines regarding the use of microorganisms in these industries. selection of microorganisms In conclusion, the for specific industrial applications is a complex process that involves a careful analysis of their metabolic capabilities, growth conditions, ease of cultivation, and safety.  2.1 Criteria for selecting microbes An ideal industrial strain should have the following qualities: The organism should be able to use wide range of low cost and easily available substrate throughout the year. The organism should produce large biomass and high amount of industrial product. The organism should be non-pathogenic and non-allergic. Organism should not degrade with passage of time i.e. it should be genetically stable. It should be easy to perform genetic manipulation on it.  Selection criteria for the biotechnological activity of microorganisms Biotechnological Microorganism Isolation source Selection criteria activity Resistance to gastric acidity and Lactobacillus and E resistance to bile salt. Adherence to nterococcus Food, plants, and mucus and/or human epithelial cells Probiotic Strains (Normal human and cell lines. Antimicrobial and microflora) antagonism activity against potentially pathogenic bacteria Saccharomyces Yeas Grapes, musts, or Fermentative power, aroma profile Wine production ts wines modulation, acidity regulation Phytotoxic Germination in pre-emergence, phytotoxicity, plant height and root secondary Fusarium fujikuroi Brazilian Pampa biome length in post-emergence, and metabolites with lesions in detached leaf-punctured herbicidal activity assay Sourdough-based Bacteria belonging Tolerance to acid, salt, sucrose, and fermentation and to the family Italian sourdoughs ethanol stresses, urease, amylase, bread production Lactobacillaceae and proteolytic activities Microbial Yeast, bacteria, and Inhibition of spore germination and antagonists fungi Varied isolation sources fungi radial growth plant pathogens  Selection criteria for the biotechnological activity Bacteria EPS precipitation using 70% ethanol Exopolysaccharide Soil samples from Al- strains. Bacillus and the bacteria colony ropy strand production Bahariya Oasis velezensis formation Recycled activated Polyhydroxybutyrate sludge [RAS] from the Gas consumption and PHB [PHB] production Methanotrophs Humber wastewater accumulation after enrichment culture from methane treatment plant situated technique in Toronto, Canada Coagulase- Traditional Chinese Hydrolysis halos revealed with Protease activity negative staphylococ fermented sausage coomassie blue and enzyme activity ci [CNS] Azotobacter vinelandii, Bacillus Level of siderophore production Siderophores megaterium, Bacillus Collection determined by chrome azurol S, CAS production subtilis, Pantoea method allii, and Rhizobium radiobacter Bacteriocinogenic Surrounding clear zone in the MRS Bacteriocin lactic acid bacteria Honeycomb filled with agar, biochemical and morphological production and Bifidobacterium oregano honey characterization spp 2.2 Microbial isolation (classical and emerging methods) Historical Perspective: From Traditional to Modern Isolation Traditional methods, like streak or pour plating, relied on spreading a sample over a growth medium and allowing individual cells to grow into isolated colonies. These techniques, while effective, often require skilled hands and considerable time. The transition to modern methods has been marked by an emphasis on precision, automation, and the ability to isolate specific microorganisms more efficiently.  Current Isolation Techniques 1. Classical Methods of Microbial Isolation a. Culture Techniques Agar Plates: – Use of solid media (e.g., nutrient agar) to isolate individual colonies from mixed populations. Streak Plate Method: A sterile loop spreads a diluted microbial sample across the surface to isolate colonies. Streak plating is a fundamental microbiology technique known for its simplicity and high effectiveness in isolating microbial colonies. The process involves delicately dragging or spreading a microbial sample across the surface of an agar plate using a sterile tool, such as an inoculation loop. As the sample is streaked, the density of the microorganisms decreases, allowing individual cells to be isolated at the end of the streaking process. This forms distinct colonies, each originating from a single microorganism or a group of the same microorganisms.  a. Culture Techniques Spread Plate Method: A diluted sample is spread evenly on the agar surface using a glass spreader. Pour Plating Pour plating is another key technique in microbiology, beneficial for quantitative studies and accommodating the growth of diverse bacterial types. In this method, a diluted microbial sample is mixed with liquefied agar and then poured over the surface of a sterile petri dish. As the agar solidifies, the bacteria are entrapped and begin to grow. Pour plating is advantageous when counting the number of viable cells in a sample is necessary, as it allows for the development of countable colonies. It also supports the growth of both aerobic and anaerobic bacteria, making it a versatile choice for various microbiological studies. This method is beneficial in applications where oxygen sensitivity of the microorganisms is a concern, as it provides an environment where surface and subsurface colonies can develop.  b. Selective Media Purpose: Media designed to favor the growth of specific microorganisms while inhibiting others. Examples: – MacConkey Agar: Selects for gram-negative bacteria and differentiates lactose fermenters. – Mannitol Salt Agar: Selects for staphylococci and differentiates mannitol fermenters. c. Enrichment Culture Technique: Use of specific growth conditions (e.g., temperature, pH, nutrients) to favor the growth of a particular microbial group. Application: Often used to isolate bacteria from complex samples (e.g., soil, water) by creating a selective environment. d. Serial Dilution Process: Serially diluting a sample to reduce the concentration of microorganisms, followed by plating to isolate colonies. Purpose: Helps in quantifying viable microorganisms in a sample.  2. Emerging Methods of Microbial Isolation a. Molecular Techniques PCR-Based Methods: Polymerase Chain Reaction (PCR): Amplifies specific DNA sequences to identify and isolate microorganisms based on genetic material. – Metagenomics: Allows for the analysis of genetic material directly from environmental samples, bypassing the need for culturing. b. Fluorescent In Situ Hybridization (FISH) Technique: Uses fluorescent probes that bind to specific microbial RNA or DNA sequences, allowing visualization and identification of microorganisms in complex samples. c. Single-Cell Isolation Methods: – Laser Capture Micro dissection: Isolates specific cells using a laser to cut them out from a tissue or mixed culture. – Microfluidics: Employs small channels to isolate and analyze individual microbial cells.  Emerging Methods of Microbial Isolation d. Culture-Independent Techniques Environmental DNA (eDNA) Analysis: – Extraction and sequencing of DNA from environmental samples to identify microbial communities without culturing. Next-Generation Sequencing (NGS): – Provides comprehensive profiles of microbial diversity and abundance in samples, aiding in the identification of microorganisms. e. Bioinformatics Tools Analysis of Sequencing Data: Bioinformatics approaches analyze sequencing data from metagenomic studies to identify and categorize microbial species based on genetic information.  Comparative Overview Method Advantages Limitations Simple, cost-effective, Not all microbes can be cultured; Classical Culture straightforward. bias towards fast-growing species. Targets specific groups, enhances May miss uncultivable or slow- Selective Media isolation. growing microbes. Enrichment Increases the chances of isolating Risk of contamination; may not Cultures specific microbes. represent natural community. Molecular Can detect and identify Requires specialized equipment Techniques unculturable microbes. and expertise. Single-Cell Allows for detailed study of Technically challenging, Isolation specific cells. expensive. Provides insights into microbial Requires computational resources Bioinformatics diversity and function. and expertise.  Challenges and Limitations Both traditional and modern techniques have limitations. Traditional methods can be labor-intensive and may not always isolate the most clinically or environmentally relevant microbes. Meanwhile, advanced technologies can be cost-prohibitive and require specialized training and equipment.  Future Perspectives The future landscape of microbial isolation techniques is poised for remarkable advancements, with research intensely focused on developing methods that are more efficient, highly selective, and scalable. Artificial intelligence and machine learning are likely to have significant impact, automating systems to identify and isolate specific microbes with exceptional precision using sophisticated algorithms, marking a significant advancement in microbiological research and analysis. This technological evolution will minimize human error and significantly enhance the speed and volume of microbial analysis. Integrating AI and machine learning in microbial isolation represents a groundbreaking shift set to transform both the accuracy and scope of microbiological research and industrial applications.  2.3 Strain improvement and maintenance Strain improvement is one element of fermentation process management. It is the process of increasing the productivity of a microorganism by improving or selecting for a more productive phenotype.  2.3.1 Methods of Strain Improvement I. Strain Improvement of Industrially Important Microorganisms It is an important tool for improving the productivity of bioprocesses. In general, there are three main strategies for improving the productivity of microbes. 1) Selection of high-yielding mutants: This approach involves screening a population of microorganisms for variants that produce more product than the parental strain. The most common method for selecting high-yielding mutants is to plate cultures on media containing a selection agent that is toxic to the parental strain but not to the mutant. High-yielding mutants can then be isolated by growing colonies from the plates. 2) Genetic engineering: This approach involves manipulating the genetic sequence of a microorganism to improve its productivity. One common strategy is to insert genes from other organisms that encode enzymes and catalyze the production of the desired product. 3) Metabolic engineering: This approach involves altering the metabolic pathways of a microorganism to redirect its resources towards the production of the desired product. One common strategy is to knock out genes that are not essential for product formation and replace them with genes from other organisms that encode enzymes that catalyze the production of the desired product.  SCREENING OF MICROORGANISMS (PRIMARY & SECONDARY SCREENING) Screening is the detection and isolation of high-yielding species of microorganisms from the natural sources material, such as soil containing a heterogeneous microbial population is called Screening. Screening can also be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from among a large microbial population. In microbial technology, microorganisms hold the key to the success or failure of a fermentation process. It is therefore important to select the most suitable microorganisms to carry out the desired industrial process. The most important factor for the success of any fermentation industry is of a production strain. It is highly desirable to use a production strain possessing the following four characteristics:  SCREENING OF MICROORGANISMS It should be high-yielding strain. It should have stable biochemical/ genetical characteristics. It should not produce undesirable substances. It should be easily cultivated on large-scale.  SCREENING OF MICROORGANISMS Thus to be effective, screening allows the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the small percentage of useful microorganisms that are present in the population. During screening programs except crowded plate technique a natural source such as soil is diluted to provide a cell concentration such that aliquots spread, sprayed or applied in some manner to the surface of the agar plates will yield well isolated colonies (30-300). The screening programs generally consists of- 1. Primary screening and 2. Secondary screening  SCREENING OF MICROORGANISMS Primary screening: It is carried out just to detect and isolate the species from mixed population that posses desired capacity. It is generally carried out by crowded plate technique. A natural source such as soil is diluted to provide cell concentration such that amount spread, applied in some manner to the surface of agar plate which will yield colonies not touching neighboring colonies. The following are some examples of primary screening- – Screening of Antibiotic producing organisms – Screening of organic acid and Amine producing organisms – Screening of organisms producing Vitamins, Amino acids and Growth Factors extracellularly – Screening of Antibiotic producing organisms: It is generally done by crowded plate technique. The diluted source is spread on the agar plate and the organisms are allowed to grow in incubator. After incubation the ability of particular colony to produce antibiotic is indicated by development of inhibition of growth around the colony.  SCREENING OF MICROORGANISMS Primary screening carried out by this technique does not necessarily select antibiotic producing organisms, because inhibition around the colony may be due to change in pH of surrounding medium by the colony and rapid utilization of nutrients in the surrounding area. Thus, further testing again is required to prove that the inhibitory activity associated with a microorganism can really be attributed to the presence of an antibiotic. The crowded plate technique has limited application, since usually we are interested in finding a microorganism producing antibiotic activity against specific microorganism and not against the unknown microorganism that were by chance on the plate in the vicinity of an antibiotic producing organism. Antibiotic screening is improved, therefore by the incorporation into the procedure of a “Test organism” that is an organism used as an indicator for the presence of specific antibiotic activity.  SCREENING OF MICROORGANISMS Dilutions of soil or of other microbial sources are applied to the surface of agar plates so that well isolated colonies will develop. The plates are incubated until the colonies are a few millimeters in diameter and so that antibiotic production will have occurred for those organisms having this potential. A suspension of test organism is then sprayed or applied in some manner to the surface of the agar and the plates are further incubated to allow growth of the test organism. Antibiotic activity is indicated by zones of inhibited growth of the organism around antibiotic producing colonies. In addition a rough approximation of the relative amount of antibiotic produced by various colonies can be gained by measuring in mm the diameters of the zones of inhibited test organism growth. Antibiotic producing colonies again must be isolated and purified before further testing.  SCREENING OF MICROORGANISMS 1. Screening of organic acid and Amine producing organisms: For this serial dilutions of soil samples are prepared and spread on the surface of the medium so that after incubation well isolated colonies which do not touch the neighbouring colonies. The medium used contains sugars and pH indicating dye like neutral red or bromothymol blue, which indicates trace quantities of acid or amine produced by organism. In another method instead of pH indicating dye, calcium carbonate may be added. The production of acid or amine by colony is indicated by zone of dissolved CaCo3.  Screening of microorganisms 2. Screening of organisms producing Vitamins, Amino acids and Growth Factors extracellularly: For isolation of such organisms medium must be totally lacking the metabolite under consideration. Again microbial source is diluted and plated to provide well isolated colonies. The test organisms must posses a definite growth requirement for particular metabolite. Such metabolites will be indicated by zone of growth or increased growth.  Secondary screening 3. Secondary screening: Primary screening is usually followed by secondary screening to test the capabilities of organisms which are obtained in primary screening. Secondary screening allows the further sorting of those microorganisms that have real value for industrial purposes and discarding valueless microorganisms. Thus the secondary screening allows determination of the most efficient and suitable organism to be used for commercial production of the fermentation product from different potential strain as detected and isolated by primary screening. Secondary screening is conducted on agar plates, in flasks or small fermentors containing liquid media. Agar plates are not as sensitive as liquid culture but have advantage in secondary screening because more information is obtained, and also agar plates take relatively little space in incubator & do not require amount of handling & workup effort associated with liquid culture. Agar culture provides only limited indication of actual product yield among various isolates. To obtain this information we must employ liquid culture because it provides a much better picture of nutritional, physical & production responses of an organism to actual fermentation production conditions.  secondary screening There are two types of secondary screening techniques: 1. Qualitative secondary screening 2. Quantitative secondary screening The qualitative secondary screening tells us the spectrum or range of microorganisms which are sensitive to newly discovered antibiotic but quantitative secondary screening tells us yield of antibiotic which can be expected when microorganisms is grown in various different media. Secondary screening should yield the type of information that is needed in order to evaluate the potential microorganisms for industrial use. These are:  Secondary screening Secondary screening determines whether the microorganisms are producing new fermentation product or not. This is determined by Paper Chromatography, TLC, HOLC, GLC etc. It gives an idea about the economic position of the fermentation process. It helps in providing information regarding the product yield potentials of different isolates. It should determine what types of microorganisms are involved & It provides information pertaining to the effect of different components of a medium. This is valuable in designing the medium that may be attractive so far as economic consideration. It should determine whether microorganisms are actually producing new chemical compound, which is not previously described. It should reveal pH, aeration and other nutritional requirements to particular microorganism. It determines the chemical stability of the product. Information about the solubility of the product in various organic solvents is made available. (Useful in product recovery operation and purification).  Secondary screening Chemical, physical and biological properties of a product are also determined during secondary screening. It reveals whether the culture is homofermentative or heterofermentative. Determination of the structure of product is done. The product may have a simple, complex or even a macromolecular structure. With certain types of products (e.g. antibiotics) determination of the toxicity for animals, plants or man are made if they are to be used for therapeutic purpose. It should reveal whether product resulting from fermentation occur in culture broth in more than one form. It should reveal whether the microorganisms are able to alter or destroy their own fermentation product. It gives information about number of products in single fermentation. It determines genetic stability of the Microbial strain.  2.3.2 Maintenance of Microbial Strains Proper maintenance of microbial strains is crucial for preserving their characteristics and ensuring consistent performance in applications. Several methods have been devised for preserving microbial cultures. None of them can be said to apply exclusively to industrial microorganisms. The method most suited to any particular organism must therefore be determined by experimentation unless the information is already available. A low rate of spontaneous mutation exists during the growth of microorganisms, about one in every 109 division. Lowering the metabolic rate of the organism will further reduce the chances of occurrence of mutations. Many methods of preservation for microorganisms have been developed. Therefore, there are two criteria for selecting a method of preservation for a given culture. They are: 1. The period of preservation desired, and 2. The nature of a culture to be preserved.  Maintenance of Microbial Strains Stock cultures are those cultures of microorganisms that are stored or maintained for future that remains unaltered. There are two types of stock cultures: (i) working stocks and (ii) Primary stocks. 1. The working stock cultures are those which are used frequently and they must be maintained in a vigorous and uncontaminated condition. These cultures are maintained as agar slants, agar stabs, spore preparations or broth cultures and they are held under refrigeration. They must be checked constantly for possible changes in growth characteristics, nutrition, productive capacity and contamination. 2. Primary stocks are cultures that are held in reserve for practical or new fermentations, for comparative purposes, for biological assays or for possible later screening programs. These cultures are not maintained in a state of high physiological activity and they are delved into only rarely. Transfers from these cultures are made only when a new working-stock is required, or when the primary stock culture must be sub-cultured to avoid death of the cells.  Maintenance of Microbial Strains There are three basic aims in maintaining and preserving the microorganisms. They are: - i. To keep culture alive ii. Uncontaminated and iii. As healthy as possible, both physically and physiologically. Following are the methods of preservations of microorganisms— 1. Serial subculture This is the simplest and most common method of maintaining microbial cultures. Microbes are grown on agar slants and are transferred to fresh media before they exhaust all the nutrients or dry out. An exception to this is aerobic Streptomyces spp. Where drying up of the medium has been found successful, provided the initial growth showed the production of serial hyphae. The drying of medium appeared to encourage good sporulation and the preserved specimen became simply a dried out strand of agar coated with spores, which remained viable for a few years at room temperature.  Maintenance of Microbial Strains 2. Microbial Preservation Methods Based on the Reduction of the Temperature of Growth (Freezing)/ By using low temperature. Agar-grown organisms are refrigerated as soon as adequate growth is attained as to preserve them. Freezing is rapidly gaining acceptance for preserving organisms because of its dual use for working and primary stock maintenance as well as its storage effectiveness for up to three years. It is useful for a wide range of organisms, and survival rates have been shown to be as good as freeze-drying in many organisms.  Maintenance of Microbial Strains Advantages of the freezing methods the methods are simple to use and require a minimum of equipment they save space as many hundreds of cultures can be stored in a small space; beads thaw rapidly and hence the method saves time, differently bead colors can represent different bacteria and so recognizing them is easy; the methods can be adapted for both aerobic and anaerobic organisms; the methods are suitable for situations or countries where power outages occur, as the freezer can remain cold for some time during power failures.  Maintenance of Microbial Strains Following are the methods used in freezing A. Preservation on agar with ordinary refrigeration (4 – 10°C) Aerobic microorganisms – Agar slants – Petri dishes Anaerobic microorganisms – Agar stabs overlaid with petroleum jelly – Overlaying cultures with mineral oil B. Preservation in Deep Freezers at about -20°C, or between -60°C and -80°C – Preservation on glass beads – Storage of agar cores with microbial growth C. Storage in low temperature liquid or vapor phase nitrogen (-156°C to -196°C) – Liquid nitrogen storage (Cryogenic storage) – Microbial Preservation Methods Based on Dehydration / Removal of water – Drying on sterile silica gel – Preservation on sterile filter paper – Preservation in sterile dry soil /Soil culture /Soil stock – Freeze-drying (drying with freezing)/ lyophilization  Maintenance of Microbial Strains Microbial Preservation Methods Based on Dehydration Just as reduction in temperature limits the metabolism of the organism, dehydration removes water a necessity for the metabolism of the organism. Drying on sterile silica gel Preservation on sterile filter paper Preservation in sterile dry soil /Soil culture /Soil stock Lyophilization or freeze-drying or drying with freezing Drying on sterile silica gel Many organisms including actinomycetes and fungi are dried by this method. Screw-cap tubes half-filled silica gel are sterilized in an oven. They are dried at 25°C, cooled and stored in closed containers containing desiccants.  Maintenance of Microbial Strains Lyophilization is the most satisfactory method of long-term preservation of microorganisms. It is universally used for the preservation of bacteria, viruses, fungi, sera, toxins, enzymes and other biological materials. The major steps involve in this techniques are: A thick cell or spore suspension is prepared in a suitable protective Using sterile techniques, this thick suspension is distributed in small quantities into glass ampoules. These ampoules are subjected to deep-freezing. Then the chilled ampoules are connected with a high vacuum system. The vacuum pump is turned on and the ampoules are evacuated till drying is complete. Freeze dried ampoules are then immediately sealed off and stored, most lyophilized cultures will remain viable for long periods (> 20 yrs. When needed, the cultures are recovered from the ampoules by suspending the lyophilized cells in a minimal amount of growth medium and then incubating.  Maintenance of Microbial Strains Advantages of lyophilization: As the ampoules are sealed there is no risk of contamination The prepared ampoules are easily stored,. There is less opportunity for the cultures to undergo changes in characteristics (i.e. they remain unchanged during storage period). hundreds of lyophilized cultures can be stored in a small storage space. Lyophilization cuts down the number of transfers.. Hence it is good to see aspects of strain improvements and maintenance of microbes, outlining methods for enhancing microbial traits and ensuring the viability and stability of microbial cultures.  Chapter 3: Microbial Fermentation technology 3.1 Types of Fermentation Fermentation is defined as a metabolic process in which an organism converts sugar into alcohol. It is an anaerobic process. But in the absence of oxygen, through alcoholic fermentation process, the pyruvic acid gets converted into ethanol and carbon dioxide. In lactic acid fermentation, it gets converted into lactate. There are two main types of industrial fermentation techniques such as Solid State Fermentation (SSF) and Submerged Fermentation (SF).  Solid State Fermentation This type of fermentation is carried out on a solid substrate such as saw dust, wheat bran and cereal grain. The solid substrates are the source of nutrients required for the growth of the microbes. This type of fermentation does not involve the usage of liquid medium (no water is required for the growth of the organism) but some moisture content is essential. Single pure cultures or mixed cultures may be used for this type of fermentation. Pretreatment of the raw material is carried out to increase the bioavailability of the nutrients for the microbes to act on them. Pretreatment techniques include soaking, boiling in water and other chemical treatments. It is mostly used for the production of fermented foods such as bread, yoghurt and cheese. The foods produced through this process are nutritious and easily digestible.  Solid State… Examples of Solid State Fermentation Production of cheese by Penicillium roqueforti Production of -amylase by Aspergillus niger Production of edible mushrooms such as Agaricus and Pleurotus. The solid substrate commonly used for this process is sawdust.  Submerged Fermentation Submerged Fermentation This type of fermentation technique involves the growth of the microorganisms as suspension on liquid medium called nutrient broth. It is also known as liquid fermentation and it utilizes substrates like molasses, soluble sugars, vegetable juices, sewage water and broths. It usually requires a high content of oxygen for the growth of the organisms and it is mostly suited for bacterial cells that require high moisture content. The microorganisms utilize the nutrients on the broth and produces bioactive compounds. The compounds are usually secreted in the fermentation broth and are harvested by different techniques which is subjected to centrifugation and finally dried and packed. It is mostly exploited for industrial production of enzymes. Examples of Submerged Fermentation – Production of citric acid by Aspergillus niger – Production of lactic acid by lactic acid bacteria  Fermentation Modes Batch Fermentation It is a closed culture system. The microorganisms are usually inoculated into a fixed volume of medium that supplies the essential nutrients for the growth of the microorganisms. As the growth phase of the microbe proceeds, there occurs depletion of the nutrients which is a disadvantage of the method. During the stationary phase, the microbes produce metabolites that are extracted from the Fermenter through down streaming processes. The accumulation of metabolites also ceases the growth of the organisms in batch fermentation.  Fed Batch Fermentation It is a modified method of batch fermentation. This fermentation is the most widely used one nowadays. In this fermentation, the substrate is added in increments several times during the fermentation process to increase the concentration of the biomass. Since the substrates are added at different periodic intervals, nutrient depletion is avoided during this process and hence the growth of the biomass is maintained at optimal level. The duration of the log and stationary phase of the growth of microorganisms is also high in this fermentation process and hence the production rate of the microbial products or secondary metabolites is also high. These are highly utilized in industrial processes.  Continuous Fermentation In this type of fermentation, fresh nutrient medium is added into the fermenter continuously or in between during the course of the fermentation process. Likewise the used medium with the microorganisms are also subsequently removed from the fermenter tank for the recovery of the microbial product. High productivity is the major advantage of this technique. Volume of the medium and concentrations of the nutrients are maintained at optimal level during the course of the fermentation process.  3.2 Fermenter A Fermenter is a device that is used to carry out the fermentation process utilizing microorganisms, which is why it is also known as a “Fermenter or Bioreactor.” It contains all of the components required for the commercial synthesis of compounds such as antibiotics, enzymes, and drinks in a variety of sectors. One of the oldest methods of food preservation is fermentation. It has changed, refined, and diversified throughout the centuries. Fermentation has remained an important technology throughout mankind’s history, despite developments in food science and technology giving rise to a wide spectrum of new food technologies. Fermentation is said to provide numerous advantages. It preserves and enriches food, promotes digestion, and improves taste and flavour. The microorganisms in the fermenter are built in such a way that a larger value of the product can be formed with a minimal amount of media. It has a wide range of applications in the food sector, wastewater treatment, and so on. Fermentation is the process of bioprocessing raw substrates using microbial biomass (mostly yeasts and bacteria).  Types of Fermenter Fermentation systems can be liquid (also called submerged) or solid (also called surface). The majority of fermenters used in industry are types of submerged fermenters, which conserve space and are easier to manage and build. Submerged fermenters come in a variety of shapes and configurations, and they can be classified as aerated or anaerobic, batch or continuous. Aerated Stirred Tank Batch Fermenters are the most often utilised fermenters. There are five different types of fermenters: – Stirred tank fermenter – Airlift fermenter – Fluidised bed fermenter – Packed bed fermenter – Photo fermenter  Stirred Tank Fermenter The motor drive shaft and a variable number of impellers make up this component (more than one). The diameter of the impellers is a third of the vessel’s diameter. This bioreactor has a height-to- diameter ratio of around 3:5. A single opening in the tube attached externally allows air to enter the culture media. It makes it possible for the contents to be distributed more evenly throughout the ship. The following are some of its primary functions: – Homogenisation – Solid-state suspension – The medium is aerated – Change of heat – Rotating stirrer and baffle can be located at the top or bottom of a stirred tank fermenter. It mostly employs a batch fermentation procedure.  Airlift Fermenter It is made out of a single container that contains a hollow tube. “Draft tube” is the name for this hollow tube. At the bottom of the fermenter is a gas flow entrance that permits oxygen to pass through. The perforated disc or tube is connected to the gas flow input, allowing air to be distributed continuously. Mechanical agitation arrangements are not present in this sort of bioreactor. The airlift name suggests that the medium is lifted aloft by the air. Internal liquid circulation channels allow the medium to move continuously. Fermentation takes place at a set volume and circulation rate in this environment. Fluidized Bed Fermenter This bioreactor has a larger top section. The fluid’s velocity is slowed by this expansion. It has a narrow bottom section. It’s made in a way that: 1. Inside the vessel, the solid remains. 2. And then there’s a flow of liquid out the other end.  Packed Bed Fermenter It comprises a cylindrical vessel and a biocatalyst-packed bed. The properties of the solid matrix employed in the packed bed fermenter are as follows: 1. Non-porous or porous 2. Extremely compressible 3. Rigid A nutrient broth constantly flows over the immobilized biocatalyst in this type of fermenter. Following that, a product is released into the fluid at the bottom of the culture jar, where it can then be retrieved. Fluid can flow upward or downward in this area.  Photo Fermenter This fermenter operates on the basis of light energy, which can be achieved through direct sunshine or artificial lighting. It’s utilised to make p-carotene, astaxanthin, and other antioxidants. Glass or plastic make up the majority of this sort of bioreactor. The following components make up a photo fermenter: 1. 1 container 2. Tubes or panels count Hence, a fermenter is a closed cylindrical tank that supports the biochemical and chemical activity of microorganisms to carry out the conversion of raw materials into usable products. Because it uses microbial biomass to complete the fermentation process, it is also known as a bioreactor. Fermentation procedures are separated into surface and submersion processes depending on the usage of stock culture. Surface fermentation involves growing biomass on the raw substrate’s surface, whereas submersion fermentation involves growing microbes in the raw substrate.  3.3 Upstream processing Upstream and downstream processing are two steps inherent to the production of active pharmaceutical ingredients (API) used in biopharmaceuticals. Upstream processing takes small quantities of engineered microbial or mammalian cell culture to grow it to larger volumes in the controlled environment of bioreactors. As the subsequent process step, downstream processing includes the separation and purification - by means of chromatography or centrifugation and filtration - of the cells generated during the upstream bio- processing step. The production and processing of APIs is challenged by increasing regulation and manufacturing costs.  What is upstream processing? As already mentioned, upstream processing in biotechnology describes the step of preparing cell cultures for fermentation. It encompasses various steps, which will be described in further detail below. The main aim of upstream bio-processing, however, is to achieve large-scale cell- growth from small amounts from a variety of cell lines. The required volume can vary,. Certain parameters, such as glycosylation patterns for monoclonal antibody (mAb) products, are primarily impacted by the upstream process and need to be monitored during the entire process development. One can differ between two types of upstream bioprocessing in a bioreactor. In perfusion, also called upstream continuous bio-processing, cell-culture is removed from the bioreactor and replaced with fresh cell-culture media continuously. It was implemented to produce modern biopharmaceuticals where time to market counts. Perfusion is performed for maximum efficiency due to high flexibility, efficient use of facilities and cost reductions which come with it. Whereas in fed batch systems nutrients are fed to the bioreactor during cultivation. Fed-batch reactions typically last up to 14 days.  Steps in upstream processing The upstream part of a bioprocess refers to the initial stage in which microbes/cells are grown from either bacterial or mammalian cell lines in bioreactors. Cultivation and cell growth can be achieved by adding nutrients, growth hormones or cell culture media to the fermenter. Microbial organisms can grow much faster than mammalian cells, with cultivation times of several days or even hours. Upstream processing involves the following steps, all of which are related to inoculum development: 1. Master cell bank (MCB) 2. Working cell bank (WCB) 3. Media Preparation 4. Cell Culture 5. Cell Separation 6. Harvest and clarification Once the cells are ready for harvesting, they will be extracted before being further processed in the downstream processing step. In the clarification filtration steps, the harvested material of a bioreactor is prepared for downstream purification by reducing the content of impurities and particles.  3.4 Downstream processing The cells have been cultivated and harvested, they need to be recovered and purified to be of further use for bio- manufacturing processes. Downstream processing implies manufacture of a purified final product - including antibiotics, hormones and enzymes - usually procured in large scales, while analytical bio-separation (also achieved by downstream processing) refers to the purification process for the sole purpose of measuring a component or components of a formulation. The latter may require sample sizes as small as a single cell, while approved vaccines and gene therapy products require larger quantities, which makes the option to scale-up not only desirable; rather, it is viewed as an integral part of process development.  Steps in downstream processing 1. Separation (Solid liquid separation) 2. Cell disruption (Release of intracellular products) 3. Extraction 4. Isolation 5. Purification 6. Polishing 7. Virus filtration/inactivation 8. Concentration  Downstream processing Typical operations to achieve the removal of insoluble’s are filtration, centrifugation, sedimentation or precipitation. Product isolation is the removal of components with properties that vary considerably from that of the desired final product. Isolation steps to remove impurities include solvent extraction, adsorption and ultrafiltration. Major Stages in DSP 1. Solid liquid separation 2. Release of intracellular products 3. Concentration.i.e to remove water to achieve product concentration. 4. Purification. 5. Formulation. i.e maintain storage and distribution. Summary of major steps in DSP Desired product in culture Cell disruption (physical, chemical, Intracellular productenzymatic Extracellular product methods) Broth with solids and liquids. Products must be purified since found mixed with undesired chemicals. Solid liquid separation Cell disintegration. (Disruption). (Floatation, flocculation, filtration, Cells can be disrupted by centrifugation). Physical method Chemical method Enzymatic methods Physical methods:- E.g Ultrasonication, osmotic shock Concentration (e.g leaving cells in 2% cucrose solution), high heat (Evaporation, liquid extraction, membrane shock (thermolysis) by heat, High pressure, filtration, precipitation, adsorption) homogenization by pressure, grinding with glass with high speed in vessel, e.t.c Alkalis, organic solvents and detergents can be use in chemical methods. Purification by chromatography Lysozyme, glucanase and protease used in (gel filtration, ion-exchange affinity, enzymatic methods. hydrophobic interaction) Formulation (drying, freeze-drying, crystallization) Final product  Optimization of Bio-processing The development and cultivation of monoclonal antibodies (mAbs), gene therapy vectors, and other advanced treatments is a highly specialized process dealing with extremely sensitive biologics, thus requiring sophisticated solutions. The optimization of bio-processing unit operations not only yields significant savings but more importantly leads to increased safety and reliability. In addition, bioreactor upgrades and the implementation of single-use technologies can achieve an increased degree of scalability that allows to take different formulations from lab to large-scale production. But in order to minimize the risk of bio contamination and product loss, optimizing biomanufacturing processes extends beyond the upgrade of upstream and downstream unit operations. The optimization of upstream and downstream processes happens on the increasing use of single-use systems. Typical stainless steel bioreactors and tubing are being replaced by smaller, more flexible single-use systems. These offer maximum flexibility, cost savings, scalability, and a small environmental footprint.  Optimization of Bio-processing Aseptic aliquotation and cryopreservation of drug substances are crucial process steps in bioprocessing. Even though they’re not in the spotlight of bio-manufacturing. In drug development and drug delivery, biologics must be transferred at numerous occasions. Along the journey of monoclonal antibodies, vaccines and more, the drug substance passes lots of process steps where liquid transfer is required. It is vital that APIs have consistent product characteristics, regardless of the production batch or container in which they were collected following final filtration. Variations in the concentration of product- and/or process-related impurities are to be prevented at all costs as they can have an impact on the efficacy of the product, and consequently the patient’s safety.  Chapter 4: Application of microbes in industrial biotechnology Microbes with desired characters are isolated in pure culture and stored in stock culture collection. Microbes can be subjected to genetic manipulation to obtain microbes with desired features to be used in industry. i.e rDNA-technology. There is a requirement for industrial fermentation to optimize conditions (temp, pH, O2-aeration) for maximum amount of product with maximum economic profit. Soya bean meal, NH4, NO3, e.t.c used as N-source. Culture of two types in bioreactors known. 1. Batch culture:- is a culture replaced totally after one cycle. 2. Continuous culture:- is a culture not replaced and continuously used by addition of nutrients.  Application of microbes While working with bioreactors (fermenters), it is important to consider 1. Aseptic handling of materials and process. 2. Sterilizing growth medium (substrates) and fermenters. 3. Techniques to maintain physical parameters to the optimum. E.g pH, temperature. 4. Controlling flow of growth limiting substances (especially in continous culture). E.g Vitamins, Minerals. 5. Methods of reverting and purifying the products.  Microbial major industrial products. Microorganisms used. Industrial products Ethanol from glucose Saccharomyces cerevisiae Ethanol from lactose Kluyeromyces fragilis Acetone and butanol Clostridium acetobutylicum Enzymes Aspergilus, Bacillus, Mucor,Trichoderma Agricultural products Gibberellins Gebberelia fujikuri Food additives Antibiotics Penicillium, Streptomyces, Bacillus, Alkaloids Claviceps purpurea Steroid transformations Rhizopus, Arthrobacter Insullin, hormones for growth, interferons, E.coli, S.cerevisiae by rDNA technology. Biofuels Hydrogen Photosynthetic microbes Methane Methanobacterium Ethanol Zymomonas Both antibiotics and organic acids are secondary metabolites (wastes) secreted by microbes  Food flavoring agents and supplements. Flavoring agents like glutamic acid and vanillin are produced using fermentation. They are produced by the addition of precursors Some microbial enzymes also used to modify flavor of foods and beverages. E.g Naringinase which are flavor and flavor enhancer.. Enzymes. Enzymes have various commercial application. E.g textiles, tanneries, food industries They are replacing conventional chemical catalyst in industries. Enzymes are economically attractive, consume less energy, require no expensive corrosion resistant equipments, and do not produce unnecessary products. Enzymes based process are ‘environmentally friendly’ as they are biodegradable. Microbes are extensively used in large-scale industrial processes to manufacture numerous products that are beneficial to humans. Microbes are instrumental in the fermentation process, leading to the production of numerous products. Fermented beverages, malted cereals, broths, fruit juices, and antibiotics are some of the common products obtained through industrial processes.  Beverages Yeasts, a type of single-celled microorganism belonging to the Fungi Kingdom, are commonly used in the production of beverages like beer, brandy, rum, wine, whiskey, etc. Specifically, the Brewer’s Yeasts, scientifically known as Saccharomyces cerevisiae, are used for fermenting fruit juices and malted cereals to produce ethanol. In Post fermentation, these beverages are distilled to produce both alcoholic and non- alcoholic beverages.  Organic Acids Microbes are also employed in the industrial production of specific organic acids. The first organic acid discovered through microbial fermentation was citric acid from lemon, a citrus fruit. Organic acids can also be directly produced from glucose. Aspergillus Niger, Acetobacter acute, and Lactobacillus are some of the microbes used for the industrial production of organic acids.  Antibiotics Antibiotics are chemical substances generated by certain microbes that kill or inhibit the growth of harmful microbes without affecting host cells. The first discovered antibiotic was Penicillin, found by Alexander Fleming in 1928 from the fungus Penicillium notatum. Several other antibiotics, including Streptomycin, are produced by microorganisms to treat various bacterial infections.  Vitamins Vitamins are organic compounds that perform numerous life- sustaining functions within our bodies. They are essential micronutrients required in small quantities for the body's metabolism. Since our bodies cannot synthesize these vitamins, they need to be supplied through our diet. Besides plants and animals, microbes can also synthesize vitamins. For instance, certain groups of microbes living in the digestive tracts of humans and other animals, collectively known as gut microbiota, are involved in synthesizing vitamin K. Other examples of microbial vitamins include ascorbic acid, beta-carotene, biotin, ergosterol, folic acid, vitamin b12, thiamine, pantothenic acid, riboflavin, and pyridoxine. Besides these products, microbes are also used in the production of biofuel, vaccines, protein, and other hormonal supplements to treat malnutrition and other deficiency diseases in both humans and animals.  Microorganisms in the food industry Lactic acid bacteria, such as Lactobacillus, Lactococcus, and Leuconostoc, are used to ferment dairy products. Vinegar is the result of bacteria in the Acetobacter genus converting ethyl alcohol to acetic acid. Lactic acid is created by Lactobacillus bacteria, which are microaerophilic bacteria. Microorganisms in the health industry A vaccine is made up of weakened or destroyed forms of the bacterium, its toxins, or one of its surface proteins and contains an agent that resembles a disease-causing bacteria. The agent induces the immune system to recognize the agent as foreign, destroy it, and “remember” it so that the immune system can recognize and eliminate any of these germs it encounters in the future. Antibiotics are made in a factory using a fermentation technique in which the source microorganism is cultured in big containers with a liquid growth medium. Microorganisms in the agricultural industry The importance of different plant-associated microorganisms is well understood, for example, the legume–rhizobium interaction and the significance of mycorrhiza in plant growth promotion. Microorganisms play an important role in the nitrogen cycle by the process of nitrogen fixation, ammonification, nitrification, and denitrification. Microorganisms in the bio-fuel industry Microbial biofuel generation is mostly accomplished by yeast fermentation of sugars to produce ethanol. In industry, Saccharomyces cerevisiae is predominantly used. A defined culture of a fermenter and/or syntropy in conjunction with an aceticlastic (acetate degrading) and hydrogenotrophic (hydrogen-consuming) methanogen could produce biogas. The inoculum for biogas production comprises microorganisms found in cow dung or wastewater sludge. Hydrogen has long been recognised as a side product of photosynthesis or as a final end product of fermentation in a variety of microbes. Biohydrogen is produced by photosynthetic microorganisms, such as cyanobacteria and green algae. Microorganisms in the mining industry Acidithiobacillus ferrooxidans is increasingly being employed to extract important metals, particularly copper, from low-grade ores that would otherwise be unworkable using traditional methods. Other metals, such as uranium and gold, are also extracted by bacteria; the methods differ slightly, but they all entail the conversion of an insoluble compound to a soluble one. Microorganisms in the water industry For the treatment and purification of dirty water, biological wastewater treatment employs a variety of bacteria and other microorganisms. The water self-purification principle is used in this process. Microorganisms in the cosmetic industry Fatty acids, enzymes, peptides, vitamins, lipopolysaccharides, and pigments found in microbes have favourable cosmetic effects. Microbes produce unique compounds, such as ceramides, mycosporine-like amino acids, carotenoids, and fatty acids, like omega-3, 6, and 9, which have a wide range of applications in the cosmetic business. Hence, we are living in an era where the importance and applications of microbiology range from those in our day-to-day life to those in industrial fields. Microbiology has proven to be essential in the health industry for the production of vaccines and antibiotics as well as in the agricultural, food, bio-fuel, and cosmetic industries.  4.1 Alcoholic beverages Brewery. Beers manufactured from grains. The starch must be hydrolyzed to fermentation sugar. The starch must be hydrolyzed to fermentable sugar, maltose, glucose prior to fermentation by yeasts. For barley, amylase used to hydrolyze it. Upon germination, large amount of amylase formed. After germination, the barely is dried, stored and it is known as malt which is dark in color, it has more flavor. The first step in beer making is grinding of malt and its suspension in water to allow hydrolysis of starch. Hydrolysis of proteins also occur as same time involving enzymes. After the saccharification reached the desired stage, the mixture is boiled and subjected to further enzymatic changes and then filtered. The filtrate called wort. A hop is added to filtrate. The hop contributes a soluble resin, which imparts characterstics bitter flavor to beers. It also acts as preservatives against growth of bacteria. Beer fermentation involves heavy inoculation with yeasts S.cerevisieae. the fermentation is allowed to continue for 5-10 days at low temperature. Generally two strains of brewery yeasts, namely bottom and top yeasts. 1. Bottom yeasts- slow fermentation best at low temperature 12-15 oC. Which produce lighter beer with low alcohol content. 2. Top yeasts:- are vigorous fermentation at high temperature (20oC) that produce heavy beers with high alcohol content. The top yeasts come up to surface to the vat during fermentation and are characterized by rapid evolution of carbondioxide. The bottom yeasts settle down to bottom with slow rate of carbondioxide evolution. Fermented brew is transferred into storage or aging tank and to the finishing tank where it combined with carbondioxide. i.e carbonating the brew. From finishing tanks, it will be barreled or bottled. Bottled beers are pasteurized to prolong the shelf life.  Winery Wine making is the fermentation of grape juice. It is done by pressing grapes into CO2 and ethyl alcohol. Most grape juice are acidic that contain 10-25% sugar by weight. In most part of the world, must is fermented by a complex succession of yeasts flora (S.cerevisieae var apiculatus) and wine yeasts (S.cerevisiae val ellipsoideus) that replace it. Fermentation product contains – Traces of alcohols than ethyl alcohol. – Acids responsible for winy taste. – Esters which creates its aroma. – Glycerin (add a touch of sweetness. Fermentation process is done vigorously and completed in few days. Usually the must is first treated with sulfur dioxide which eliminate natural yeasts flora and then inoculated with desired strain of wine yeasts.  Winery Cooling fermenting mixture controls a rate of fermentation to prevent rise of temperature that affects quality of wine or that may kill yeasts. White grapes------------------ give white wine. Red grapes ------------------ give red wine. After fermentation, new wines are stabilized and are aged to produce final product. Final process may require months and for high quality red wines, even years. Especially red wines undergo a second phase of fermentation by action of LAB (Lactobacillus, Leuconostoc, or Pediococcus) during first year of aging. This process produces the acidity of wine which gives good quality red wine. E.g Champagne.  How is Potable Alcohol (Alcoholic Beverage) obtained? All alcohol or alcoholic beverages are obtained by a process called fermentation. It is concentrated or increased in strength by distillation. The percentage of alcohol in a drink varies from 0.5 – 9.5%, depending on the method by which the alcohol is obtained. Fermentation is the process in which the yeast acts on sugar and converts it to ethanol and gives off carbondioxide. The fermented iquid has 3-14% alcohol and it can be concentrated up to 95% by a series of distillations. Distillation is the process of separating elements in a liquid by vaporization and condensation. In the distillation process,the alcohol which is present in the fermented liquid is separated from water.  1. Fermented Beverages Fermented beverages can be divided into two groups, wines and beers,broadly defined. Wines are fermented from various fruit juices containing fermentable sugars. Beers come from starch- containing products, which undergo enzymatic splitting by diastase, malting, and mashing,before the fermentable sugars become available for the yeasts and bacteria. In short, wines are fermented and beers are brewed and fermented. Wines can be defined as alcoholic beverage made from fermented grape juice.  Brewed and Fermented Drinks It is similar to fermented drinks but the only difference is that the base ingredient, which is usually malted and crushed cereal, is brewed in hot water to extract maximum soluble sugar from the malt. It is then cooled, and allowed to ferment with the addition of yeast. Example–Beer and Sake Beer is classified on the basis of type of fermentation– Top or Bottom. Top fermented beers are known as Ales while bottom fermented beers are knownas Lagers.  2. Distilled Beverages Too btain any alcoholic beverage fermentation is required and the strength of alcoholis increased by application of distillation. These drinks are distilled from a base of a fermented liquid and have a high percentage of alcohol compared to fermented drinks. Newly distilled spirit (young spirit) is raw, sharp and harshin taste. These drinks are aged in wooden barr