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

Sugars

•Biofuels•Biomaterials•Biochemicals• Primary

metabolite• secondary

metabolite

The IB Value Chain

BiofuelsH2

Ethanol

SugarsFeedstocks- Renewable- Fossil

BiochemicalsFood IngredientsPharmaceuticalsFine Chemicals

BiomaterialsPolylactic acid

1,3 propane diolPHAs

Bioprocesses

Bulk

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:

1. Degradation of polysaccharides to fermentable sugars;

2. fermentation; and

3. 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 TechnologiesFirst 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

- Oil crops (e.g. rapeseed, oil palm, soy, canola, jatropha, castor, …)- 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

Biofuel type Specific name Feedstock Conversion Technologies

Bioethanol Cellulosic bioethanol Lignocellulosic biomass and biowaste

Advanced hydrolysis & fermentaion

Biogas SNG (Synthetic Natural Gas) Lignocellulosic biomassand 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)

Lignocellulosic biomassand residues

Gasification & synthesis

Biohydrogen Lignocellulosic biomass and biowaste

Gasification & synthesis or biological process

*Use GMO as a feedstock to facilitate hydrolysis / technologies for hydrogen production

Overview of Biofuel Production TechnologiesSecond/Third* Generation Biofuels

Biofuel transformation processes

First generation

Second generation

ETBE: Ethyl tetra butyl Ether

Fatty Acid Methyl Ester

Fatty Acid Ethyl Ester

Biofuel uses

• Bioethanol– 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.

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 .

Sources of Enzymes

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).

Enzyme Sources Application

a-Amylase Aspergillus E Baking

Catalase Aspergillus I Food

Cellulase Trichoderma E Waste

Glucose oxidase Aspergillus I Food

Lactase Aspergillus E Dairy

Lipase Rhizopus E Food

Rennet Mucor miehei E Cheese

Pectinase Aspergillus E Drinks

Protease Aspergillus E Baking

E: extracellular enzyme; I: intracellular enzyme

Fungal Enzymes

Enzyme Sources Application

a-Amylase Bacillus E Starch

b-Amylase Bacillus E Starch

Asparaginase Escherichia coli I Health

Glucose isomerase Bacillus I Fructose syrup

Penicillin amidase Bacillus I Pharmaceutical

Protease Bacillus E Detergent

Bacterial Enzymes

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

VACCINES

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.