Introduction to Biotechnology

• Biotechnology is the controlled use of biological agents for beneficial purposes.
• Biotechnology includes using living organisms, cells, or components to produce things or improve living things for humans.
• Classical biotechnology includes fermentation for products like alcohol, beer, wine, dairy, vinegar, citric acid, amino acids, and vitamins.
• Modern biotechnology is similar to classical biotechnology in using living organisms, but it uses new techniques.
• Modern techniques in biotechnology allow precise manipulation of biological processes at the cellular and molecular level.
• Genetic engineering allows transferring a single gene's property from one organism to another.
• A new branch of biotechnology called bioinformatics and computational biology has emerged through the use of information technology and the internet, particularly in genomics and proteomics.
• Biotechnology is applied in various fields such as food, agriculture, medicine, and solving environmental problems.
• The four major industrial areas of biotechnology application are health care (medical), crop production and agriculture, non-food uses of crops (e.g., biodegradable plastics, biofuels), and environmental uses.
• Biotechnology is divided into industrial, agricultural, medical/pharmaceutical, and environmental areas.

Industrial Biotechnology

• Industrial biotechnology (or microbial biotechnology) uses microorganisms to get valuable products or activities on a commercial scale.
• Microorganisms used in industrial processes can be natural, lab-selected mutants, or genetically engineered strains.
• Industrial biotechnology adapts and modifies biological systems in nature to produce goods and services.

Bioprocess Technology

• A bioprocess is an industrial application of biological reactions mediated by cells, microorganisms, or enzymes under controlled conditions.
• Bioprocesses transform raw materials into products that can be food, medicine, or industrial compounds.
• Bioprocesses can detoxify industrial wastes or treat factory effluents (with or without byproduct).
• Converting a lab-scale biochemical reaction to an industrial bioprocess involves biochemical engineering.
• Bioprocess technology involves upstream processes, bioreaction and bioreactors, and downstream processing.

Importance of Microbial Culture

• Microorganisms like bacteria and fungi are cultured to obtain products beneficial for humans like antibiotics, ethanol, and enzymes.
• Biotechnology and genetic engineering use microorganisms to produce recombinant molecules like insulin, hepatitis B vaccine, growth hormones, and interferons.
• Microorganisms can be cultured for biomass production (single cell protein, SCP).
• Scientists culture microbes for research purposes to understand how microbes produce important molecules in laboratories or industries.

Factors Affecting Microbial Culture

• A microbial culture is like a factory where microbe metabolism converts raw materials into products.
• Each microbial cell has a maximal capacity to convert raw materials into products over time.
• To produce a specific product quantity, the number of cells and doubling time must be calculated.
• Microbes require appropriate temperature, pressure, pH, solvent, nutrients (growth medium), and substrate (raw material).
• Requirements for culturing microbes differ between lab and industrial scales.

Types of Microbial Culture

• Microbial systems can be cultured in different ways, depending on the microbial system type or desired product.
• The same organism can produce different products by changing nutritional and other parameters or culturing vessels.

Batch Culture

• Batch culture is a closed system with a limited initial nutrient amount.
• After inoculation, the organism grows through lag, log (exponential), stationary, and decline phases.
• Growth leads to nutrient consumption and excretion of microbial products.
• At the stationary phase, growth is zero.
• In batch culture, cells experience a changing environment due to nutrient consumption and metabolite accumulation.

Fed-Batch Culture

• Fed-batch culture involves continuously or sequentially feeding a batch culture with fresh medium without removing the growing culture.
• In fed-batch systems, the substrate concentration remains constant, and the cell density increases over time.
• In fed-batch systems, the volume in the culture vessel increases over time.

Continuous Culture

• Continuous culture provides a continuous supply of microbial growth/products.
• The growth medium is designed with a limited amount of one nutrient.
• Exponential growth continues until the limited nutrient is exhausted; then, growth stops.
• Fresh medium containing the limited nutrient is added just before it is fully exhausted.
• This cycle is then repeated every time the limited nutrient is about to exhaust.

Microbial Culture or Bioprocess Techniques

• Upstream processes, bioreaction and bioreactors, and downstream processing are the main parts of bioprocess technology.

Upstream Processing

• Aseptic treatment of substrates or raw materials with the microorganism or biocatalyst is done in upstream processing.
• Upstream processing includes isolation and purification of microorganisms, media preparation and sterilization, feeding into the bioreactor, regulation of temperature/pH/pressure, and aseptic inoculation of cell culture.

Microorganisms' Isolation

• Culturing microbes is primarily for producing useful products.
• Microbes that produce substances of interest must be found through research.
• Microbes widespread in natural habitats, including soil, aquatic environments, and extreme conditions like arctic waters and hot springs, are sources for isolation.
• Desired traits of microbes can be improved after their isolation using a variety of methods.

Sources of Industrial Microbial Strain

• Isolation from the environment and culture collections are the sources of industrial microbial strains.

Isolation from the Environment

• Shotgun and objective approaches are adopted for isolating suitable industrial microorganisms from the environment.
• The shotgun approach is the collection samples of microorganisms from different material and habitats, with isolates screened for traits.
• The objective approach considers desired characteristics as components of natural microflora, sampling sites contaminated by the material an organism should degrade.

Culture Collections

• Culture collections receive and distribute deposits of strains for research and development.
• Culture collections provide a rich source of microorganisms that are of past, present and potential future interest.
• There are around 500 culture collections globally, which may be small and specialized or large notably national collections.
• The American Type Culture Collection (ATCC) holds all types of microorganisms.
• The functions of culture collections are to maintain the existing holdings, collect new strains, and provide pure culture samples.
• Using microorganisms from culture collections saves costs compared to environmental isolation.
• A disadvantage of culture collections is that competitors have access to the same microorganism.

Downstream Process (Isolation of Microbial Products)

• Once fermentation is done, the desired metabolite is recovered.
• Downstream processing involves separation of cells from the fermentation broth, purification, of the metabolite and cell disruption.
• The steps of downstream processing are separation of cells, cell disruption (if intracellular), initial purification, metabolite-specific purification, and polishing of the metabolite (98-100% purity).
• Important steps are separation of cells, concentration, metabolite-specific purification and final purification.

Microbial Metabolites

• Trophophase describes the log or exponential phase of a culture when the sole products of metabolism are essential, such as amino acids, nucleotides, proteins, carbohydrates, or ethanol, acetone, butanol.
• Primary metabolites are those synthesized during the trophophase and are the byproducts of energy-yeilding metabolism.
• Idiophase describes the phase of culture when products other than primary metabolites are synthesized and do not have a role in cell metabolism.
• Secondary metabolites are produced during the idiophase, and are synthesized from the intermediates and end- products of primary metabolism.
• Secondary metabolites are not common to the vast majority of microorganisms.

Testing Microbial Metabolites for Bioactive Activity

• For anti-microbial activity: Metabolites of the test organism are tested against test organisms.
• Soil suspension/particles are placed on agar seeded with the test organism(s), clearing zones are isolated, purified, and studied.
• For enzyme inhibition: Microorganisms are tested for producing drugs against certain disease, measuring product or unreacted substrate using spectrophotometry.
• Testing is also done for morphological changes in fungal test organisms.
• Pharmacologically active products in broth can affect spore germination or hyphal morphology.
• Animal tests are conducted on microbial metabolites to determine the content of useful drugs in the broth.

Induction of the Secondary Metabolites' Overproduction

• Methods to induce the overproduction of secondary metabolites are empirical and include mutations and stimulation by manipulating conditions.
• Mutations are: Naturally occurring variants are subjected to mutations and selected randomly.
• The effects of precursors are that production is stimulated.
• Penicillin production was stimulated by phenylacetic acid in corn steep liquor.
• Inorganic compounds phosphate levels encourage growth, which negatively impacts secondary metabolite production while manganese specifically encourages idiophase production, particularly in bacilli.
• The temperature range that permits good growth is around 25°C among microorganisms, while lower temperatures, around 5-10°C, encourage secondary metabolite production.

Improvement of Industrial Strain

• Strain isolation only identifies strains that can produce a molecule, not that they can produce enough of it.
• Classical genetics and genetic engineering techniques improve desirable strain characteristics.
• Strain improvement is done with genetic recombination to combine genetic elements from two genomes.
• Mutagenesis is another strategy.
• Screening and selection identify strains suited to industrial fermentation processes.

Techniques Used in Strain Improvement

• Natural recombination and mutagenesis are the techniques used in strain improvement.

Natural Recombination

• Bacterial DNA is in the form of a single chromosome and plamids that contains supplemental genetic information.
• Bacteria exchange genetic material through processes of conjugation, transduction and transformation.

Mutagenesis

• Mutations result in physical changes to the DNA of a cell.
• Repeated mutagenesis with selection and screening improves industrial microorganisms.
• Mutants can be naturally-arising or induced.
• Mutation is induced by exposing industrial strains to chemical (e.g. nitrosoguanidine or NTG) or physical (e.g. UV rays) mutagens and selecting mutants with improved characteristics.
• Multiple successive mutations are necessary before obtaining desired results.

Practical Isolation of Mutants

• There are three stages before a mutant can come into use: exposure to a mutagen, exposure to conditions to select mutant, and the testing phase.

Exposing Oragnisms to the Mutagen

• Strain is exposed to chemical or physical mutagens, mutants with improved characteristics selected.
• Multiple mutations are carried out before desired results are reached.
• A classic strain example is improving antibiotic penicillin: where several successive mutations were necessary to develop Penicillum.

Methods for Isolation of Microbial Mutants

• Direct Observation, Enrichment Technique and Replica-Plating Technique are the techniques used in isolation of microbial mutants.

Direct Observation Technique

• Observation of the normal type and observation of non-pigmented colonies indicate mutants.
• Indicators can be incorporated into media to detect microorganisms.
• PH indicators detect the production of acidic products

Enrichment Technique

• Enrichment isolates mutants resistant to phages, antibiotics and toxic chemicals.

Replica-Plating Technique

• Replica plating transfers colonies from one plate to another to compare growth of same colonies of different media to determine conditions.
• One needs to test antibiotic resistance to test bacterial strains and nature of mutation.
• Isolates nutritional mutants and antibiotic resistant mutants.
• Replica plating involves selectively growth media inoculated with microorganisms from original plate, replicating the original spatial pattern of colonies.
• The purpose of replica plating is to be able to compare the master plate and any secondary plates, to screen for phenotype.

Auxotrophic Mutants

• Auxotrophic mutants lack the enzymes to manufacture required nutrients, which must be added to the growth medium.
• Wild-type possess all the enzymes to synthesize growth requirements.
• Auxotrophic mutants are in industrial microbiology to product amino acids and nucleotides.
• Organism is transferred from slant to broth of medium, broth grown on medium and transferred in complete medium.

Modern Tools in Strain Improvement (Genetic Engineering Techniques)

• Genetic engineering techniques adds new properties and capabilities to microorganisms.
• Using the techniques, the microorganisms are manipulated to synthesize and secrete enhanced of biomolecules, produce novel compounds, and utilize cheaper substrates.
• Human are often created in microbes using these techniques.

Making Recombinant DNA

• Recombinant DNA involves purifying vectors, and containing the genes to be cloned.
• Vector DNA is first digested with suitable restriction endonuclease and made liner with or without sticky ends.
• Isolate DNA with same restriction and vector.
• Linearize vector and target DNA cut and are incubated together in presence of DNA ligase during incubation.
• A phosphodiesterlinkage is established between them, mediated by the DNA ligase enzyme.
• Cells inserted into host cells selected and cells carrying vector selected.

Vectors: The Vehicle for Cloning

• Vectors must have certain features such as an origin of replication, a selective marker, a unique restriction site, and be small in size so entry and transfer is easy to the host cell.

• Plasmids: Plasmids are extrachromosomal, self-replicating, usually circular, double-stranded DNA molecules found in bacteria and some yeasts.

• Plamsids are important to growth in cells or growth and often confer useful properties to the host such as antibiotic resitances which are selective.

• Plasmids can be 1 to 2 copies or multiples copy inside the host organism.

• Plasmid vectors described can only replicate in E.coli and many of vectors that in Eukaryotic cells can exist in both E.coli.

• Shuttle Vectors contain two types of origin of replication and selectable maker, one functions in eukaryotic cells and E.coli.