MICROBIAL PROCESSING

BIOTECHNOLOGY

Dr Roshini Govinden

UKZN – Dept Microbiology

PRE-REQUISITEMICR 213rDNA202

AIMSTo introduce key concepts and applications in microbial processing

Range and scope of microbial processing

Growth of microbes in a controlled environment:

Nutrient requirements

Kinetics

Environmental parameters

Monitoring

Batch and continuous processes

Aspects of upstream and downstream processing

Examples of major fermentation processes

Current trends and applications in industrial biotechnology

Regulatory, safety (HACCP) and socioeconomic considerations ofbiotechnology

CONTENTS

To describe the applications of microbes in industry

To describe the design of bioreactors for different applications

To design media, growth conditions and techniques for producing andrecovering different types of products of commercial value

To recognize how to screen for, preserve and propagate important genotypes

To compare and contrast between different fermentation processes

To apply kinetic formula to determine growth and productivity parameters ofbatch and continuous fermentations

To describe the role of recombinant DNA technology in industrialbiotechnology

Learning Objectives

To construct a HACCP plan

To describe the use of microbial cells and enzymes in immobilized forms

To be familiar with several industrial production processes involving microbes

Practicals

Isolation of antibiotic and enzyme producing strains

Downstream processing techniques

Production of yeast biomass in batch culture & batch culture kinetics

Production of food via fermentation techniques - yogurt, beer

4 theory lectures per week – including one tutorial every fortnight

1 practical per week

Assignment

Self Study

Attendance will be monitored for all lectures and tutorials

DP Requirement

40% Class Mark

80% attendance at practicals

Assessment

Class Mark (40%)Theory tests 20% x 2 = 40%

Assignment 10% x 1 = 10%

Practical Reports 15%

Pre-pract quizzes 5%

Practical Tests 15% x 2 = 30%

Total 100%

3 hour exam (60%)

TEST DATES

THEORY TESTS

2 September 2014

30 September 2014

PRACTICAL TESTS

19 August 2014

7 October 2014

LECTURER

Dr R Govinden

Room F3 03 018

Consultation Times

Monday: 8H30-10H00

Wednesday: 8H30-10H00

LABORATORY TECHNICIAN

Ms B Mbatha

TUTORS

Ms A Arjoon

Ms R Singarum

• Microbial Processing: use of microbes and/or their

enzymes to produce products useful to industry

• Biotechnology is ….the manipulation of living

organisms or their constituents, particularly at the

molecular genetic level, to form products useful to

industry

What is Biotechnology?

How does it work?

Multidisciplinary•Biology

•Microbiology

•Biochemistry

•Molecular biology

•Genetics

•Chemistry

• Engineering

Traditional biotechnology:

conventional techniques

used for many centuries to

produce beer, wine, cheese

etc.

“New” biotechnology: all

methods of genetic

modification by

recombinant DNA

technology and modern

developments of

“traditional” biotechnology

Time line of Biotechnology

• 10 000-8000 BC

– Humans domesticate crops and livestock.

– Potatoes first cultivated for food.

– Wine was known in the Near East by the time

of the book of Genesis (10 000 BC)

• 6000-4000 BC

– Sumerians and Babylonians were drinking beer by 6 000 BC

– Yeast first used to leaven bread by 4 000 BC (Egypt)

– Production of cheese and fermentation of wine (Sumeria, China & Egypt)

• 4000 BC— 2000 BC

– Babylonians control date palm breeding by selectively pollinating female

trees with pollen from certain male trees.

• 500 BC

– First antibiotic: mouldy soybean curds used to treat boils (China)

• 1322

– An Arab chieftain first uses artificial insemination to produce superior horses

• 1590

• Janssen invents the microscope.

• 1663

• Hooke discovers existence of the cell.

• 1675

- Leeuwenhoek discovers bacteria.

• 1797

- Jenner inoculates a child with a viral vaccine to

protect him from smallpox.

• 1830 – 1833

- 1830 — Proteins discovered.

- 1833 — First enzyme discovered and isolated.

• 1857

• Pasteur proposes microbes cause fermentation.

• Demonstrated the fermentative ability of

microorganisms 1857 and 1876 – the father of

biotechnology.

• 1859

• Charles Darwin publishes the theory of evolution by

natural selection.

• The concept of carefully selecting parents and culling the

variable progeny greatly influences plant and animal

breeders in the late 1800s despite their ignorance of

genetics.

• Modern fermentation industry owes its existence to the First

World War (1914-1918).

– Glycerol : manufacture of explosives.

• byproduct of soap manufacture from the First World

War

• yeast to produce glycerol by fermentation of sugar

• Neuberg’s Method

• advantage of simultaneous production of alcoholic

substances for industrial use.

Industrial microbiology - result of empirical developments in the

production of wine, vinegar, beer and sake, and with the

traditional fungal fermentations used in Asia and Africa for the

production of food.

experimental approach to the production of microbial

metabolites only began at the beginning of the 20th century.

Up until the time of World War II, the main microbial products

that had developed from this experimental approach were

enzymes such as proteases, amylases and invertase.

• breakthrough in biochemical and microbial engineering-after

World War II - large-scale production of the first antibiotic,

penicillin.

• engineering developments

• techniques for large-scale sterilization, aeration & growth of

microorganisms.

• genetic methods - microbial strain improvement

Introduction of sterility to biotechnological processes:

1940s, complicated engineering techniques were applied to the mass cultivation of microorganisms to exclude contaminating microorganisms, for production of

antibiotics

amino acids

organic acids

enzymes, steroids

• 1942• The electron microscope used to identify and characterize

a bacteriophage

• Penicillin mass-produced in microbes.

• 1944• DNA is proven to carry genetic information — Avery et

al.

• Waksman isolates streptomycin, an effective antibiotic

for tuberculosis.

• 1953

– The scientific journal Nature publishes James Watson and

Francis Crick's manuscript describing the double helical

structure of DNA, which marks the beginning of the modern

era of genetics.

• From World War II up until about 1960

– Major new biotechnology products were antibiotics.

• numerous new antibiotics were discovered

• 20 were put into commercial production

– chemical transformation of steroids.

– culture of animal cells for the production of virus vaccines

• 1960 -1975

– in Japan, new microbial processes for the production of flavour

enhancers

• amino acids

• 5’-nucleosides

– Processes for enzyme production for industrial, analytic and

medical purposes were perfected.

– During this same period, successful techniques for the

immobilization of enzymes and cells were developed.

• 1972

– The DNA composition of humans is discovered to be 99

percent similar to that of chimpanzees and gorillas.

continuous fermentation

production of Single-Cell Protein (SCP) from yeast and

bacteria for use as human and animal food.

SCP processes were developed using microorganisms capable

of using petroleum-based starting materials such as gas oil,

alkanes, and methanol.

microbial biopolymers such as xanthan and dextran, used as food

additives, were also developed into commercial processes.

use of microorganisms for tertiary oil recovery (an aspect of geo-

microbiology)

perfection of techniques for anaerobic cultivation of

microorganisms - derived out of studies on the sewage treatment

process.

• 1975 –

– development of the hybridoma technique, for the production

of monoclonal antibodies

• 1977

– First expression of human gene in bacteria - E. coli.

• 1978

• Recombinant human insulin first produced.

• 1980

– The U.S. Supreme Court, in the landmark case , approves the

principle of patenting recombinant life forms, which allows the

Exxon oil company to patent an oil-eating microorganism

• 1982

– The first product, human insulin, was soon followed by Factor

VIII, human growth hormone, interferons and urokinase.

• 1983

– The polymerase chain reaction (PCR) technique is conceived.

PCR, which uses heat and enzymes to make unlimited copies of

genes and gene fragments, later becomes a major tool in biotech

research and product development worldwide.

– The first genetic transformation of plant cells by Ti plasmids is

performed.

• 1990

– The first experimental gene therapy treatment is performed

successfully on a 4-year-old girl suffering from an immune

disorder.

– The first transgenic dairy cow - used to produce human milk

proteins for infant formula - is created.

• 1994

– First FDA approval for a whole food produced

through biotechnology: FLAVRSAVR™ tomato.

The birth of a lamb (Dolly) cloned by nuclear transfer using an

mammary cell from an adult sheep as nucleus donor and

an enucleate ovum as recipient.

1998

– A rough draft of the human genome map is produced, showing the locations of more than 30,000 genes.

2000

– First complete map of a plant genome developed: Arabidopsis thaliana.

• containing a fluorescent

marker gene from a jellyfish.

• 224 eggs were injected with

the gene.

• yielded 40 viable embryos

on fertilization

• 5 pregnancies resulted.

• 3 monkeys born live

• only ANDi carries the gene,

although it is not being

expressed.

Birth of first genetically

modified primate, a rhesus

monkey named ANDi

(inserted DNA backwards)

DIFFERENT ASPECTS OF MICROBIOLOGY

• Industrial Microbiology

• Food Microbiology

• Environmental Microbiology

• Medical Microbiology

• Pharmaceutical Microbiology

INDUSTRIAL MICROBIOLOGY

Industrial Application of Microbiology &

Biotechnology

INDUSTRIAL

• AMINO ACID PRODUCTION

• ANTIBIOTIC PRODUCTION

• ALCOHOL PRODUCTION

• DETERGENT PRODUCTION

Microbes are

used in the

production of

vaccines and

antibiotics e.g.

Penicillin from

Penicillium sp

HEALTH CARE PRODUCTS

Antibiotics Against PathogensAntibiotics Against

Pathogens

41

Some important fermentation products

Product Organism Use

Ethanol Saccharomyces

cerevisiae

Industrial solvents,

beverages

Glycerol Saccharomyces

cerevisiae

Production of

explosives

Lactic acid Lactobacillus

bulgaricus

Food and

pharmaceutical

Acetone and

butanol

Clostridium

acetobutylicum

Solvents

-amylase Bacillus subtilis Starch hydrolysis

42

Some important fermentation products

FOOD AND DRINK

•MANUFACTURE• Cheese

• Yoghurt

• Wine

• Beer

FOOD PRODUCTS

AND MICROBIOLOGY

The yeast S.cerevisiae

used in bread making

and beer brewing

FOOD AND BEVERAGES

Fungi in cheese

making e.g. Penicillium

camembertii

Edible fungi e.g.

Agaricus sp.

(mushrooms)

INDUSTRIALLY IMPORTANT MICROBIAL

METABOLITES

• CITRIC ACID PRODUCTION – soft

drinks

• PLANT HORMONES –

GIBBERELLINS – AGRICULTURAL

INDUSTRY

• VITAMINS – RIBOFLAVIN

INDUSTRIALLY IMPORTANT

MICROBIAL ENZYMES

Bread making and confectionary

Dairy industry

Detergents

Cosmetic industry

Glucose syrup production

Improving quality and flavour of

food

Pulp and paper industry

AGRICULTURAL APPLICATIONS

FERTILIZER WASTEWATER TREATMENT

PROCESSES

Industrial Manufacturing Processes

• biotechnology offers us many options for minimizing the

environmental impact of manufacturing processes

• by decreasing energy use

• and replacing harsh chemicals with biodegradable

molecules produced by living things.

Renewable Energy

• Fossil fuels are nonrenewable.

• Gradual move toward a biobased economy in which agricultural

operations will be the energy and natural resource fields of

tomorrow.

ENVIRONMENTAL

Water testing and purification processes

Wastewater treatment processes

Environmental Microbiology

Bioremediation

Bio-leaching

Bio-mining

Microbial – enhanced oil recovery and petroleum upgrading

Mineral and Energy Recovery

Biofuel, Bioethanol and Biomass Production

BIOTECHNOLOGY TECHNIQUES

• Fermentation

• Cell Fusion

• Cell/tissue Culture

• Genetic Engineering

Fermentation:

Technique that requires bioreactors - allow a biological process to

take place under optimum conditions, producing a useful compound

in large amounts.

Simple bioreactors have been used to produce beer and wine.

Some bioreactors carry out a chemical process without using living

cells

enzymes are used to trigger conversion of one chemical into another by a

process called biotransformation.

corn syrup and vitamin C

Cell Fusion:

combining two cells to make a single cell that contains the

genetic material of the original cells.

create new plants by fusing cells from species that do not

naturally hybridise and then generating new plants from the

fused cells.

Pomato (potato/tomato), rutabaga (turnip/wild cabbage),

brokali (brocolli/kale), aprium (plum/apricot), loganberry

(blackberry/red raspberry), lemato (lemon/tomato)

also used to make fused cells known as monoclonal antibodies,

- protective proteins produced by a clone of a single cell.

Liposomes:

• used as vehicles for delivering certain drugs to the target

tissues of the body.

• Drugs are encapsulated in liposomes - protection against

digestive enzymes in the stomach.

• Liposomes are microscopic spherical particles that are formed

when lipids form a suspension in the water.

Cell and Tissue Culture:

• growth of living cells or organisms outside the body in a

suitable culture medium which provides nutrients to the

growing cells.

• Individual cells grow and divide in a sterile medium.

• used extensively in laboratories, e.g., cancer research, plant

breeding and for routine analysis of chromosome karyotypes.

Genetic Engineering:

technology requiring rDNA technology and gene transfer

techniques to bring about a change in the genetic constitution

of the organism to express desired traits.

Previously animal and plant breeders employed classical

breeding methods to improve

produced economically important plants, cows, horses, dogs

Techniques for isolating and altering genes were developed by

geneticists in the US in the 1970s by manipulating the genome

of bacteria.