1

Introduction to MicrobiologyIntroduction to Microbiology

CHNG 3804 CHNG 3804

Biochemical EngineeringBiochemical Engineering

Fariba DehghaniFariba Dehghani

Introduction

• Biochemical Engineering is an extremely broad field.

• Products range enormously in value. – Pharmaceuticals

– Sewage treatment

• An understanding of Biochemical Engineering is useful in many traditional Chemical Engineering Fields – Mining: Acid Mine Drainage & Bio-Leaching.

– Fouling of Heat Exchangers, Bacterial growth in Cooling Water.

Biochemical Engineering

• Multidisciplinary subject

• Concerned with developing biological processes on an industrial scale.

Chemistry Biochemistry

Biotechnology

MathematicsEngineering

Microbiology

Genetics

Pharmacy

Food Technology

Biochemical engineering is not:Biochemical engineering is not:

• Genetic engineering

• The Human Genome Project

• Genetically modified crops

• Gene therapy

• Stem cell research

What do we do?What do we do?

CELLS

mammalian

plant

insect

fungal

bacterial

viral

gen

etic

mo

dif

icat

ion

(bio

)rea

cto

r

sep

arat

ion

PRODUCTS

proteins

acids

alcohol

clean water, soil

clean air

biogas

Biochemical Engineering Process

ProductformulationRaw materials Bio-reactor

Process Control

Bio-catalystSelection andManipulation

ProductSeparation and

Purification

2

Bio-catalyst Selection and Manipulation

The following are critical for selection of biocatalyst

• It should be a pure culture,

• Be genetically stable,

• Be easily propagated,

• Exhibit rapid growth characteristics,

• Have good rate of product formation,

• Be free of toxic byproducts

• Be amenable to genetic manipulation.

Applications of Biochemical Engineering

• Pharmaceuticals – Antibodies, antigens, porcine and human growth hormones interferon,

tissue plasminogen activator, diagnostic and other therapeutic compounds

– Therapeutic peptides, cytolytic toxins, nasal decongestant

• Specialty chemicals– Amino acids, vitamins, biopolymers, enzymes, lipids

• Foods and beverages– Sweeteners, alcoholic beverages, single cell protein

• Environmental Engineering– Bioremediation of soil, industrial effluents, and waste water treatment

• Commodity chemicals– Acetic acid, acetone, butanol, ethanol, etc.

• Bioelectronics– Biosensors, biochips

Classical microbiological processesClassical microbiological processes

• Micro-organisms transformed foods:

– Alcoholic beverages (beer, wine, spirits –yeast)

– Bread (yeast)

– Dairy products (yoghurt, cheese – lactic acid bacteria)

– Vinegar (acetic acid bacteria)

– Soybeans (yeast, lactic acid bacteria)

Industrial microbiologyIndustrial microbiology

• Ethanol (yeast)

• Glycerol (yeast)

• Acetone (bacteria)

• Butanol (bacteria)

• Isopropanol (bacteria)

• Butanediol (bacteria)

• Lactic acid (bacteria)

• Citric acid (fungi)

AntibioticsAntibiotics

• Penicillin (fungi)

New microbiological processesNew microbiological processes

• Carotenoids and steroids (fungi)

• Amino acids, nucleotides (bacteria)

• Enzymes (amylase, proteinases, pectinases)

• Recombinant proteins (hormones, antigens, antibodies)

3

Process designProcess design

• Biochemical process design requires knowledge of the capacity of cell cultures (biomass) to make products

• Products may be secreted externally or stored in inclusion bodies in the cell

• Profitability ($) can be determined by:

– How much product is recovered per gram of biomass?

– How much substrate is consumed per gram of biomass?

– How much product is recovered per gram of substrate consumed?

Microbiology

• To study Biochemical Engineering requires some knowledge of Microbiology.

• Humans have used microorganisms for a very long time.

• Bread and cheese using yeasts

• Beer Brewing may have begun 7,000 BC.

• Microorganisms complete critical segments of carbon, nitrogen and oxygen cycles.

• Microorganisms are also responsible for many human, animal and plant diseases.

What are Bacteria

• Unicellular entities

• Neither plant nor animal

• Diverse lifestyles

• Highly adaptable, metabolic and biosynthetic machine

• Different cell structure to other macro cells making up higher animals and plants

Significance of Bacteria

• Global scale: carbon, nitrogen, phosphorous and sulfur cycling

• Spoilage of foods

• Pathogenic to plants and animals

• Production of high value compounds, amino acid, alcohol, antibiotics, food supplements

• Used in composting

Modern Microbiology

• Schleiden and Schwann 1838

All living systems are composed of cells and their products.

• In the late 1800’s Pasteur identifies microorganisms as critical active agents for fermentation and develops the germ theory of disease.

Classification of Microorganisms

PROTIST KINGDOM

PROCARYOTES EUCARYOTES

Bacteria Blue Green Algae Fungi Algae Protozoa

Molds Yeast

4

Procaryote

• Do not contain a membrane enclosed nucleus.

• Relatively small and simple cells.

• Typically 0.5 – 3 µm.

• Can grow very fast.

• Usually exist alone.

• May be spherical, rods or spirals.

• Cell is surrounded by a rigid wall.

• Large Surface area to volume ratio.

Salmonella Pseudomonas

Microstructure of Bacteria

Dillow et al., Nature, 1998

S. aureus

E.coli Bacteria

• Gram Negative and gram positive

• Spore forming bacteria

• Important Industrial Examples

– Lactic Acid Bacteria: Production of Yoghurt.

– Rhizobium: Biological Nitrogen Fixation.

– Genetically Engineered Escherichia coli: used widely for the production of Proteins.

The cell structureThe cell structure

Microbiology text book

Gram positive and Gram Negative bacteriaGram positive and Gram Negative bacteria

• Gram positive: thick peptidoglycan layer

• Gram Negative: Thin peptidoglycan layer

• Peptidoglycan composed of two sugar derivatives: N-

acetylglucosamine (NAG) and N-acetylmuramic acid

(NAM)

• A small peptide containing D- and L-Alanine, D-

glutamic acid and either L-Lysine or m-diaminopimelic

acid

5

Gram positive and Gram Negative bacteria

Peptidoglycan

Plasma membrane

Outer

membrane

Peptidoglycan

Plasma

membrane

Microbiology text book

Spore

Wall

Cortex

Endospores are resistant to temperature, desiccation, chemical agents, and enzyme. Autoclaving required to use high temperature to inactivate spores.

Microbiology text book

Blue Green Algae (Cyano Bacteria)

• Not used to great commercial benefit.

• Important in Aquatic Systems and Nitrogen Cycle.

Eucaryote

• 1000 to 10,000 times larger than Procaryotes.

• All cells of higher organisms belong to this family.

• Greater degree of spatial organization and differentiation.

• Slower growing than Procaryotes.

• Slower rates of mass transfer due to smaller surface area to volume ratio.

• Some are shear sensitive – particularly animal cells.

Microstructure of the Cells

Cell wall

Plasma membrane

Ribosome

DNA

Nucleus

Prokaryotic cell

Typical animal cell

Typical plant cell

Campdell, 1999

Saccharomyces cerevisiae

http://www.mycolog.com/chapter6.htm

6

Yeasts

• One of the important subgroup of Fungi

• Single Small Cells 5 – 30 µm.

• Saccharomyces Cerevisae grown aerobically for use a bakers yeast.

• A number of varieties are grown anaerobically to convert sugar to ethanol.

• Saccharomyces strains used anaerobicallyfor beer and wine production.

MoldsMolds

• Higher form of fungi.• Growing structure called mycelium.• Grow in branched structures.• Mold and yeast do not contain

chlorophyll• This has rheological, mass transfer

and growth implications.• Examples:

– Penicilium that is used for production of antibiotics

– Aspergillus niger that produces oxalic acid (HO2CCO2H) and citric acid

• Cheese ripening

Algae and ProtozoaAlgae and Protozoa

• Algae are of research interest for conversion of CO2 into fuel.

• Algae is used as food supplement such as seaweed.

• Protozoa can not exploit sunlight energy, and are significant in biological waste treatment.

Animal and Plant CellsAnimal and Plant Cells

• Large and Slow growing.

• Shear Sensitive.

• Animal cells are widely used for vaccine production.

• Animal cells can be used to produce proteins.

• Human cells can be grown for therapeutic use.

VirusesViruses

• A non-cellular biological entity that can reproduce only within a host cell.

• Viruses consist of nucleic acid (DNA or RNA) covered by protein; some animal viruses are also surrounded by membrane.

• Inside the infected cell, the virus uses the synthetic capability of the host to produce progeny virus.

PhagesPhages

• Bacteriophage or phage are small viruses which infect bacteria.

• Specific to the bacterial strain.

• Can be a problem in large scale pure culture bacteria plants.

• Have made essential contributions to molecular genetics.

• Used to maintain stable DNA segments.

• Used in Russia and Eastern Europe to treat infections.

7

Further ReadingFurther Reading

“The Birth of Penicillin, and the disarming of microbes” written by Ronald Hare, and published in 1970 is a very readable eyewitness account of both the discovery and large scale production of penicillin.

Fermenter Technology Fermenter Technology

Polarographic DO probes

Model based optimal control

Fluidized bed and air lift systems.1970-mid

1990’s

Computer monitoring and control;

Mathematical modelling for control of

yeast fermentations

Higher pressures to minimize DO

limitations

1960-1970

Steam sterilisable pH and dissolved

oxygen electrodes.

Feedback control

Mechanically aerated vessels, aseptic

operation,

Continuous sterilisation of feeds

1945-1960

Temperature Control,

pH electrodes used for offline

measurement/control.

Fed-batch cultures introduced to

overcome dissolved oxygen

limitations.

Steel vessels introduced for acetone-

butanol (first aseptic fermentation)

Sparging of air for yeast

Mechanical stirrers in small vessels

1900-1945

Thermometer

Hydrometer

Heat Exchangers

Wooden or copper vats

barrels, shallow trays, trickle filters and

clay pots

Pre-1900

Control EmployedEquipment UsedEra

Recombinant DNARecombinant DNA

• The term “Recombinant DNA” refers to the combination of DNA from two

different sources.

• Recombination of DNA allows the variation and diversity of living

organisms.

• The joining of the male’s sperm cell with the female’s egg cell is a natural

recombination process. The DNA of the male joins and mixes with the

female’s DNA resulting in an offspring cell with a combination of both

parents. This is evident in the offspring’s physical appearance, which

exhibits features that are similar to both parents.

• Natural recombination is now mimicked by biotechnologists allowing the

reconstruction of DNA molecules in test tubes. In this process, the

construction of a new DNA molecule takes place from two different

sources. The techniques and procedures associated with this are known

as recombinant DNA technology and are the specialty of the geneticist

(refers to “gene cloning”).

Development of Recombinant DNADevelopment of Recombinant DNA

• In 1973 Cohen, Chang, Helling and Boyer

demonstrated that a recombinant DNA molecule

can be maintained and replicated in E.coli

• Boyer founded the first genetic engineering

company Genetech soon after.

• Recombinant DNA technology allows cell to be

altered to make them more productive and to

use cells to make foreign proteins.

Gene CloningGene Cloning

Prescot LM, Harley JP and Klein DA, 1996, Microbiology, The McGraw-Hill Companies, Inc. USA

8

Recombinant DNA Technology in Recombinant DNA Technology in

BiotechnologyBiotechnology

•Custom design of products with desirable characteristics,

•Increase of a yield of a particular product,

•Improve the efficiency of the production with respect to energy utilisation,

•Transfer of a particular activity to a more desirable host

•Minimize the purification steps required.

•Feasibility to introduce mammalian genes into bacteria for large scale

production that is advantageous for pharmaceutical industry for production

of insulin and vaccines.

The overall efficiency of the biotechnology process is improved with the aid of the new recombinant techniques. The benefits of recombinant DNA technology to the biotechnology process lie in the bio-catalyst selection stage and include

Recombinant DNA TechnologyRecombinant DNA TechnologyMany human genes have been cloned in E. coli or in yeast.Cultured cells (E. coli, yeast, mammalian cells) transformed with human genes are being used to manufacture:•insulin for diabetics

•factor VIII for males suffering from haemophilia A

•factor IX for haemophilia B

•human growth hormone (GH)

•erythropoietin (EPO) for treating anaemia

•three types of interferons

•several interleukins

•granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant

•tissue plasminogen activator (TPA) for dissolving blood clots

•adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID)

•angiostatin and endostatin for trials as anti-cancer drugs

•parathyroid hormone

SummarySummary

• This Lecture

– Introductory Microbiology

– Penicillin and the Birth of Biochemical Engineering

• Next Lecture

– Introductory Biochemistry

Text Book ReferenceText Book Reference

• Any Microbiological text book

• Bailey and Ollis (1986) Chapter 1

• Campbell M. K., Biochemistry. Saunders College publishing Harcourt Brace College Publisher,1999

• http://learn.genetics.utah.edu/units/basics/

• http://en.wikipedia.org/wiki/Dna