2Industrial biotechnology Application of biotechnology for industrial purposesManufacturingAlternative energy (bioenergy)BiomaterialsIt includes the practice of using cells or components of cells like enzymes to generate industrially useful products.Division of IBIndustrialPharmaceutical biotechnology.Growing fungus to produce antibiotics, e.g. penicillin from the penicillium fungi.
3Important Applications Production of primary metabolites (Acids & Alcohol)Secondary metabolites (Antibiotic)Production of whole microbial cells (Food, Vaccine)Biotransformation reactions (Enzymes, steroids)Exploitation of metabolism (Leaching and wastes treatment)Recombinant proteins (Therapeutic proteins, gene delivery vectors, etc.)
7Some definitionsBioenergy is energy of biological origin, derived from biomass, such as fuelwood, livestock manure, municipal waste, energy crops.Biofuels are fuels produced from biomass, usually of agricultural origin.BioethanolBiodieselBiogasEnergy crops are crops specifically cultivated to provide bioenergy, mainly biofuels but also other forms of energy.e.g. miscanthus, eucalyptus.
8Liquid FuelsEthanol production by fermentation of carbohydrates is rather expensive and is influenced by theYield of ethanolEthanol tolerance of fermenting organismEthanol is relatively toxic to microbes.. Limited conc. Can accumulateEthanol-tolerant strain
10FUEL ETHANOL FROM BIOMASS Energy can be extracted from biomassby direct combustion (Common Method) orby first converting the biomass to another fuel (ethanol, methanol, or methane) and then combusting it.Cellulose, hemicelluloses, and starches are a vast renewable source of sugars convertible to ethanol by microbial fermentation.Production of ethanol From the polysaccharides of biomass proceeds in three stages:Degradation of polysaccharides to fermentable sugars;fermentation; andalcohol recovery.Disruption of the physical structure of lignocellulose makes cellulose and hemicelluloses accessible to enzymatic attack. Disruption is done bySteam explosionAcid hydrolysis
11Production of Alcohol (S. cerevisiae) Preparation of MediumAddition of water to molasses to decrease sugar conc to %.Addition of acid to adjust pHAddition of yeastAdjustment of temperatureThorough mixing of yeast inoculum with molassesFermentationVigorous fermentation leads to production of CO2, a by product of alcohol industryCollection of CO2Separation of ethyl alcoholRemoval of unused substances of molassesSeparation from other impuritiesPurificationPurification with the help of rectifying columns
12Production of Alcohol (Z. mobilis) Zymomonas mobilis, a bacterium isolated from fermenting sugar-rich plant juices, produces ethanol up to 97% of the theoretical maximum value.The advantages of Z. mobilis over S. cerevisiae with respect to producing bioethanol:higher sugar uptake and ethanol yield (up to 2.5 times higher)lower biomass productionhigher ethanol tolerance up to 16% (v/v),does not require controlled addition of oxygen during the fermentation,amenability to genetic manipulations.
13DisadvantagesIn spite of these attractive advantages, several factors prevent the commercial usage of Z. mobilis in cellulosic ethanol production.Substrate Limitation: Utilize only glucose, fructose and sucrose.Wild-type Z. mobilis cannot ferment C5 sugars like xylose and arabinose which are important components of lignocellulosic hydrolysates.Unlike E. coli and yeast, Z. mobilis cannot tolerate toxic inhibitors present in lignocellulosic hydrolysates such as acetic acid and various phenolic compounds.Concentration of acetic acid in lignocellulosic hydrolysates can be as high as 1.5% (w/v), which is well above the tolerance threshold of Z. mobilis.
18Biofuel uses Bioethanol Biodiesel Used as neat ethanol (E95, blend of 95% ethanol and 5% water)Used as E85 (85% volume ethanol with petrol) in flex-fuel vehiclesUsed as blend smaller than 5% volume (E5) in ordinary petrol or as its derivative ETBEBiodieselCurrent maximum 5% in diesel blends, otherwise can only be used in modified diesel engines
20Secondary metabolites Secondary metabolites have no function in the growth of the producing cultures (although, in nature, they are essential for the survival of the producing organism), functioning as:(1) sex hormones;(2) Antibiotics(3) ionophores;(4) competitive weapons against other bacteria, fungi, amoebae, insects and plants;(5) agents of symbiosis etc.Microbially produced secondary metabolites are extremely important for health and nutrition.AntibioticsOther medicinalsToxinsBiopesticidesAnimal and plant growth factors
21AntibioticsThe best-known group of the secondary metabolites are the antibiotics.Their targets includeDNA replication (Actinomycin)Transcription (Rifamycin)Translation (Chloramphenicol, tetracycline, erythromycin and streptomycin)Cell wall synthesis (cycloserine, bacitracin, penicillin, cephalosporin and vancomycin)
22Enzyme productionThe production of enzymes by fermentation was an established business before modern microbial biotechnology.However, recombinant DNA methodology was so perfectly suited to the improvement of enzyme production technology that it was almost immediately used by companies involved in manufacturing enzymes.Important enzymes are proteases, lipases, carbohydrases, recombinant chymosin for cheese manufacture and recombinant lipase for use in detergents.Recombinant therapeutic enzymes already have a market value of over US$2 billion, being used for thromboses, gastrointestinal and rheumatic disorders, metabolic diseases and cancer.They include tissue plasminogen activator, human DNAase and Cerozyme.
23Sources of EnzymesBiologically active enzymes may be extracted from any living organism:Of the hundred enzymes being used industrially,- over a half are from fungi- over a third are from bacteria with the remainder divided between animal (8%) and plant (4%) sources .
25Sources f EnzymesMicrobes are preferred to plants and animals as sources of enzymes because:They are generally cheaper to produce.Their enzyme contents are more predictable and controllable.- Plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants).
26E: extracellular enzyme; I: intracellular enzyme Fungal EnzymesEnzymeSourcesApplicationa-AmylaseAspergillusEBakingCatalaseIFoodCellulaseTrichodermaWasteGlucose oxidaseLactaseDairyLipaseRhizopusRennetMucor mieheiCheesePectinaseDrinksProteaseCatalase:catalyzes the decomposition of hydrogen peroxide to water and oxygen.E: extracellular enzyme; I: intracellular enzyme
27Bacterial Enzymes Enzyme Sources Application a-Amylase Bacillus E Starchb-AmylaseAsparaginaseEscherichia coliIHealthGlucose isomeraseFructose syrupPenicillin amidasePharmaceuticalProteaseDetergentAsparaginase:(EC ) is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid.Penicillin amidase: Sakaguchi and Murao1 reported on the presence of an enzyme in the mycelium of Penicillium chrysogenum and Aspergillus oryzae which would split penicillin G (I) into phenylacetic acid (II) and 'penicin' (III) :
28Therapeutic ProteinsRecombinant protein plays a big role in the creation of therapeutic agents that could modify and repair genetic errors, destroy cancer cells, treat immune system disorders, etc.For instance, Erythropoietin, a protein hormone produced by recombinant technology can be utilized in treating patients with erythrocyte deficiency, which is a common cause of kidney complications.
29Categorization of FDA approved PTs based on mechanism of action PTs replacing a protein that is deficient/abnormalPTs augmenting an existing pathwayPTs providing a novel functionPTs that interfere with a molecule/organismPTs that deliver other compounds/proteinsProtein vaccinesProtein diagnostics
31New Generation of Vaccines: Recombinant DNA technology is being used to produce a new generation of vaccines.Virulence genes are deleted and organism is still able to stimulate an immune response.Live nonpathogenic strains can carry antigenic determinants from pathogenic strains.If the agent cannot be maintained in culture, genes of proteins for antigenic determinants can be cloned and expressed in an alternative host e.g. E. coli.
32DNA VaccinesDNA vaccines are possibly the most hopeful and powerful alternative to traditional vaccines.A genetically engineered vaccine is already widely used against the liver infection hepatitis B.
33Production of Vitamin C Humans, as well as other primates, guinea pigs, the Indian fruit bat, several species of fish, and a number of insects, all lack a key enzyme that is required to convert a sugar, glucose, into vitamin C.No single bacterial genus or species is known that will carry out all of the reactions needed to synthesize vitamin C.Two species (Erwinia species and Corynebacterium genus) can perform all but one of the required steps.In 1985 a gene from one of these genus (Corynebacterium) was introduced into the second organism (Erwinia herbicola), resulting in a new bacterial form.This engineered organism can be used to produce a precursor to vitamin C that is converted via one chemical reaction into this essential vitamin.