Industrial Biotechnology

Industrial biotechnology Application of biotechnology for industrial purposes Manufacturing Alternative energy (bioenergy) Biomaterials It includes the practice of using cells or components of cells like enzymes to generate industrially useful products. Division of IB Industrial Pharmaceutical biotechnology. Growing fungus to produce antibiotics, e.g. penicillin from the penicillium fungi.

Important 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.)

Cell factories Biofuels Biomaterials Biochemicals Primary metabolite secondary metabolite Sugars

The IB Value Chain Bulk Biofuels Sugars Feedstocks Biomaterials Ethanol Sugars Feedstocks Renewable - Fossil Biomaterials Polylactic acid 1,3 propane diol PHAs Biochemicals Food Ingredients Pharmaceuticals Fine Chemicals Bioprocesses Fine

Bioenergy

Some definitions Bioenergy 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. Bioethanol Biodiesel Biogas Energy crops are crops specifically cultivated to provide bioenergy, mainly biofuels but also other forms of energy. e.g. miscanthus, eucalyptus.

Liquid Fuels Ethanol production by fermentation of carbohydrates is rather expensive and is influenced by the Yield of ethanol Ethanol tolerance of fermenting organism Ethanol is relatively toxic to microbes.. Limited conc. Can accumulate Ethanol-tolerant strain

Main bioenergy feedstocks Wood Forest management residues Fuel timber Crops Annual (cereals, oilseed rape, sugar beet) Perennial (miscanthus, reed canary grass, short rotation coppice) Wastes Straw Animal manure

FUEL ETHANOL FROM BIOMASS Energy can be extracted from biomass by direct combustion (Common Method) or by 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; and alcohol recovery. Disruption of the physical structure of lignocellulose makes cellulose and hemicelluloses accessible to enzymatic attack. Disruption is done by Steam explosion Acid hydrolysis

Production of Alcohol (S. cerevisiae) Preparation of Medium Addition of water to molasses to decrease sugar conc to 30-40 %. Addition of acid to adjust pH Addition of yeast Adjustment of temperature Thorough mixing of yeast inoculum with molasses Fermentation Vigorous fermentation leads to production of CO2, a by product of alcohol industry Collection of CO2 Separation of ethyl alcohol Removal of unused substances of molasses Separation from other impurities Purification Purification with the help of rectifying columns

Production 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 production higher ethanol tolerance up to 16% (v/v), does not require controlled addition of oxygen during the fermentation, amenability to genetic manipulations.

Disadvantages In 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.

Bioenergy crops

Overview of Biofuel Production Technologies First Generation of Biofuels Biofuel type Specific name Feedstock Conversion Technologies Pure vegetable oil Pure plant oil (PPO), Straight vegetable oil (SVO) Oil crops (e.g. rapeseed, oil palm, soy, canola, jatropha, castor, …) Cold pressing extraction Biodiesel Biodiesel from energy crops: methyl and ethyl esters of fatty acids Biodiesel from waste Waste cooking/frying oil Cold and warm pressing extraction, purification, and transesterification Hydrogenation Bioethanol Conventional bio-ethanol Sugar beet, sugar cane, grain Hydrolysis and fermentation Biogas Upgraded biogas Biomass (wet) Anaerobic digestion

Conversion Technologies Overview of Biofuel Production Technologies Second/Third* Generation Biofuels Biofuel type Specific name Feedstock Conversion Technologies Bioethanol Cellulosic bioethanol Lignocellulosic biomass and biowaste Advanced hydrolysis & fermentaion Biogas SNG (Synthetic Natural Gas) Lignocellulosic biomass and residues Pyrolysis/Gasification Biodiesel Biomass to Liquid (BTL), Fischer-Tropsch (FT) diesel, synthetic (bio)diesel Lignocellulosic biomass and residues Pyrolysis/Gasification & synthesis Other biofuels Biomethanol, heavier (mixed) alcohols, biodimethylether (Bio-DME) Gasification & synthesis Biohydrogen Gasification & synthesis or biological process *Use GMO as a feedstock to facilitate hydrolysis / technologies for hydrogen production

Biofuel transformation processes First generation ETBE: Ethyl tetra butyl Ether Fatty Acid Methyl Ester Second generation Fatty Acid Ethyl Ester

Biofuel 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 vehicles Used as blend smaller than 5% volume (E5) in ordinary petrol or as its derivative ETBE Biodiesel Current maximum 5% in diesel blends, otherwise can only be used in modified diesel engines

Manufacturing factories

Secondary 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. Antibiotics Other medicinals Toxins Biopesticides Animal and plant growth factors

Antibiotics The best-known group of the secondary metabolites are the antibiotics. Their targets include DNA replication (Actinomycin) Transcription (Rifamycin) Translation (Chloramphenicol, tetracycline, erythromycin and streptomycin) Cell wall synthesis (cycloserine, bacitracin, penicillin, cephalosporin and vancomycin)

Enzyme production The 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.

Sources of Enzymes Biologically 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 .

Enzyme Production

Sources f Enzymes Microbes 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).

E: extracellular enzyme; I: intracellular enzyme Fungal Enzymes Enzyme Sources Application a-Amylase Aspergillus E Baking Catalase I Food Cellulase Trichoderma Waste Glucose oxidase Lactase Dairy Lipase Rhizopus Rennet Mucor miehei Cheese Pectinase Drinks Protease Catalase:catalyzes the decomposition of hydrogen peroxide to water and oxygen. E: extracellular enzyme; I: intracellular enzyme

Bacterial Enzymes Enzyme Sources Application a-Amylase Bacillus E Starch b-Amylase Asparaginase Escherichia coli I Health Glucose isomerase Fructose syrup Penicillin amidase Pharmaceutical Protease Detergent Asparaginase:(EC 3.5.1.1) 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) :

Therapeutic Proteins Recombinant 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.

Categorization of FDA approved PTs based on mechanism of action PTs replacing a protein that is deficient/abnormal PTs augmenting an existing pathway PTs providing a novel function PTs that interfere with a molecule/organism PTs that deliver other compounds/proteins Protein vaccines Protein diagnostics

New 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.

DNA Vaccines DNA 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.

Production 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.