BABS1202 Study Notes - Harry Cheng

Lecture 3: Biofuels
Biomass to Energy

Energy Density of Fuels

Advantages of Liquid Fuels for Transportation
● High energy density
● Longer range
● Vehicle not too heavy = fewer acceleration losses, rolling losses
● Vehicle not too large = fewer aerodynamic losses.
● Quick and easy to refuel.
● Easy to handle.
● Easy to transport.
● Existing infrastructure

Biofuels
● Fuels derived directly from biomass
● Produced from biomass or bio-derived substrates using biological or
chemical/biochemical processes
● Produced from biomass or bio-derived substrates using thermochemical
processes
● With respect to net carbon emissions, these fuels have the common feature that
they are utilising recently “captured” carbon (from photosynthetic processes)
rather than prehistorically captured carbon reserves

Ethanol
● Formed by fermentation of sugars or by hydrolysis of ethane
● Neat (i.e. pure) ethanol is an excellent fuel
- High octane > 100 allows higher compression ratio and hence higher
efficiency
● Ethanol has a lower energy density than regular gasoline. This means the fuel
economy is lower measured in km/L.
● In cold weather, starting problems may be experienced because the vapour
pressure is too low at low temperature.
- Can be solved by direct injection or fuel heater.
● Formaldehyde emissions may be increased. Potential problems with corrosion

Ethanol as a Blended IC-engine Fuel

● Ethanol is typically uses as a blend (E5 to E10 in Australia)
- Up to E80 elsewhere (Brazil)
● Mixing properties with gasoline are thus important. Two main varieties,
depending on water content
- Ethanol azeotrope: ~96% ethanol, ~4% H2O cannot be mixed with
gasoline due to phase separation.
 - Anhydrous ethanol: >99% ethanol, can be mixed with gasoline; mixtures
e.g. E10 = 10% ethanol, 90% gasoline
● If blended fuel, the engine must be able to run on pure gasoline, i.e. advantage of
high compression ratio is lost.

Biodiesel - FAME
● FAME – fatty acid methyl ester
● Formed from transesterification of soybean or rape seed oil using a catalyst and
methanol
● Glycerol produced as a by-product
● Loss in power cf. Diesel of 5-7%
● Mixes with normal diesel (B10, B20) or used pure (B100)
● Lower emissions of particulates and CO when combusted in a diesel engine

1st, 2nd and 3rd Generation Biofuels

1st Generation Ethanol

● Basic idea is to produce ethanol by fermentation of monosaccharides (i.e. simple
sugars).
● Yeasts such as Saccharomyces cerevisiae, S. uvarum or Schizomyces pombe
(or in some cases bacteria such as Zymomonas mobilis) metabolize glucose:
C6H12O6 → 2 CH3CH2OH + 2 CO2. (Ethanol)
● Starches can also be used…
- Like cellulose, starches are polymers of glucose (C6H10O5)n.
- However, they are more branched and the bonds are weaker.
 - Starches can be broken down into glucose by enzymes or acids in a
pretreatment step called hydrolysis (i.e. adding H2O), then fermented

1st GE Process
● Basic process known for a long time, many 1000’s of years.
● Ethanol as a major automobile fuel is more recent.
- Significant interest in Brazil and USA
● For fuel-scale ethanol, steps are:
- Pretreatment such as milling
- For starch-based crops, enzymatic or acid hydrolysis to release
monosaccharides
- Fermentation
- Product extraction including distillation & dehydration (removing water
content as yeast only produces alcohol at 10% up to 15% v/v

EROEI
● EROEI = Energy Returned On Energy Invested.
● Often discussed in relation to bioenergy.
● Energy is consumed in farming (machinery, fertiliser), transportation, processing.
EROEI = energy in fuel / energy expended in production
● Needs to be >1!
● Very dependent on crop, climate, land quality, farming practice, transport
practice, etc.
● HENCE -> ALL BIOFUELS ARE NOT EQUAL

Cellulosic Ethanol
● Observation: first generation ethanol uses only sugars and starches, which are a
small fraction of plant material.
- Therefore: Large areas of land are required (also, water, fertiliser, etc.)
- There is direct competition with food..
● Observation: cellulose and hemicellulose, which comprise the major fraction of
plant matter ~70% are also both made of sugars.
● Second generation idea: exploit cellulose and hemicellulose fraction
● Pretreatment:
- Cell wall structure must first be opened up to allow enzymes access for
hydrolysis.
- They must: open structure without degrading basic sugars, and without
forming products that inhibit fermentation (e.g. furfural.), and without
leading to prohibitive costs, etc.
● Methods: acid hydrolysis, steam explosion, ammonia fiber expansion.
 ● Next step is cellulose treatment yield C6 and C5 sugars
● Overall process:

● Potential advantages (relative to first generation ethanol):

- Lower cost raw materials
- Greater availability (agricultural/forestry residues; new crops)
- no direct competition with food crops
- greater GHG reduction compared to starch-based crops
● Potential disadvantages:
- Largely unknown technology at scale: commercial scale plants yet to be
demonstrated..
- Cannot use lignin fraction (contrast to thermochemical routes).
- Sustainability issues remain: soil nutrients, use of land.

1st and 2nd Generation Ethanol Processes

Biodiesel
● Feedstocks include: Soybean oil, Canola, Palm oil, Jatropha, Waste vegetable
oils and Animal fats (tallow, lard).
● Rudolf Diesel in 1900, ran his original engine on peanut oil!
● Since then, interest in these alternative oil resources has followed oil prices.
● Biodiesel is a product resulting from the upgrading of raw straight vegetable oil
(SVO) in a transesterification reaction.
● Transesterification involves the reaction with an alcohol (usually methanol or
ethanol), in the presence of a catalyst (usually a base – NaOH or KOH)
● If the process is done carefully, a high quality fuel can result, that can be burned
in an unmodified engine, but requires a source for methanol (or ethanol) as well
as oils.

● Advantages:
- Fuel can be burned in an unmodified engine, and blended with fossil fuel.
Widely available (domestically)
- Generally low emissions
- Low net CO2 (depending on feedstock)
- Low CO & unburned hydrocarbons
- Low particulates
- When produced from waste, avoided disposal costs & environmental
impact
● Disadvantages:
- Low yield of fuel/ha
- large use of land (+water, fertilisers)
- insufficient total amounts available.
- large energy inputs per unit energy output (typical EROEI 3)
- Direct competition with other uses (food)
- Demand for oil, in part for the biodiesel industry, has led to massive
deforestation in South East Asia (Palm oil), some in Brazil (Soy bean)
- Net CO2 emissions plus loss of biodiversity
- Slightly increased NOx
Other Biofuels
● Biobutanol
● Gasification of biomass
- Syngas to methanol
- CO + 2H2 → CH3OH
● Pyrolysis of biomass
● Anaerobic fermentation products
- Biogas (methane) from fermentation
- Biohydrogen

Summary and Conclusions

● Demand for liquid fuels will continue.
● Biofuels are a form of stored solar energy and have the potential to be
renewable.
● Biofuels are categorized into:
- First generation technologies including biodiesel and ethanol that are
established and cheap but use large land areas and competing with food.
- Second generation technologies that are not yet fully commercialised but
potentially use smaller land areas and compete less with food.
- Third generation technologies, where non-arable land is utilised and
source does not compete with the food chain
● Major technologies to watch for second/third generation biofuels are:
- Cellulosic ethanol
- BTL fuels via biomass gasification including: methanol, DME, and
Fischer-Tropsch liquids
- Biohydrogen
Lecture 4: Microbial Growth
Growth Limitations
● Bacteria are incredibly diverse; and:
● Each bacterial species can grow in only a limited set of environments.
● Each bacterial species can grow only if presented with the right nutrients and
conditions.
● Bacteria produce characteristic byproducts (such as waste products).
● We use their growth characteristics to identify bacteria phenotypically

Eukaryotic vs Bacterial Chromosome

Microbial Death
● Programmed cell death
● Not as well studied as in eukaryotes
● Different types: Necrosis, autophagic cell death and apoptosis
● For the good of the bacterial community. Eg. stress response, development,
genetic transformation, and biofilm formation.
Growth Influencing Factors
● Physical Factors (extrinsic or environmental):
- pH Temperature
- Oxygen concentration
- Water activity
- Pressure
● Biochemical or Nutritional Factors (intrinsic)

Obligate Aerobes
● Dependent upon the presence of O2 as the
final electron acceptor in respiration

Obligate Anaerobes
● Cannot tolerate the presence of O2 ; dies in its
presence and uses energy producing
metabolic pathways other than aerobic
respiration (either fermentation and/or
anaerobic respiration).
● Do not possess enzymes (superoxide
dismutase, catalase) responsible for
breakdown of toxic by-products (superoxide,
hydrogen peroxide) of aerobic respiration.

Facultative Anaerobes
● Do not require the presence of O2, but do grow better when it is present (can use
aerobic respiration).
● In the absence of O2, organism uses fermentation (some organisms may use
anaerobic respiration).
● In the absence of O2, organism uses fermentation (some organisms may use
anaerobic respiration)

Aerobic Respiration
● Aerobic respiration includes redox reactions: fuel is oxidised resulting in reduced
cofactors.
● The electrons are transferred to the electron transport chain and ultimately
passed onto oxygen.
● This process powers the production of ATP
Anaerobic Respiration
● Anaerobic respiration is respiration without oxygen.
● It uses an electron transport chain, with inorganic molecules other than oxygen
as the final electron acceptor.
● These terminal electron acceptors have smaller reduction potentials than oxygen
- less energy is released per oxidized molecule.
● Anaerobic respiration is less efficient than aerobic.
● Anaerobic respiration should NOT be confused with fermentation.

Fermentation
● Fermentation only yields a net of 2 ATP per
glucose molecule while aerobic respiration
yields ~32 molecules of ATP per glucose
molecule.
● Fermentation products contain potential
chemical energy (they are not fully oxidized),
but cannot be metabolized further without the
use of oxygen
Endospore Formation
● An endospore is a dormant, resilient,
nonreproductive structure.
● Aids survival of bacterium through periods of
environmental stress.
● Dipicolinic acid is a spore specific chemical that
appears to help in the ability for endospores to
maintain dormancy. This chemical comprises up to
10% of the spore's dry weight.
● Produced by some bacteria Eg. some Bacillus and
Clostridium.
● Comprises bacterium's DNA, ribosomes and
dipicolinic acid.
● Germination = return to the vegetative state from the
endospore state.

Bacterial Growth Media

● Most commonly used:
● Luria nutrient broth – tryptone, yeast extract and salt.
● Luria nutrient agar – tryptone, yeast extract, salt and agar.
● Agar is a complex polysaccharide isolated from red algae. It is solid at room
temp, liquefies at boiling (100˚C), does not re-solidify until it cools to 42˚C.
● Agar provides a framework to hold moisture & nutrients.

Enriched Media
● Enriched media contains nutrients required to support the growth of a wide
variety of organisms.
● Commonly used to harvest as many different types of microbes as are present in
the specimen.
● Addition of complex components as required by fastidious microorganisms.

Selective Media
● Selective media are used for the growth of only select microorganisms.
● Contains one or more agents that inhibit growth of some microbes and
encourage growth of the desired microbes
Differential Media
● Differential media (sometimes called indicator media) distinguishes one
microorganism type from another growing on the same media.
● Allows growth of several types of microbes and displays visible differences
among desired and undesired microbes.

What is Growth?
● A cell can reproduce itself.
● Macroorganisms:
- Growth and reproduction can be distinct for individuals
- Growth is increase in size
- Reproduction is increase in number
● Microorganisms:
- Growth and reproduction is the same thing
- Growth is an increase in the number of cells

Growth Curve
● The growth curve represents
the increase, decrease or no
change in the number of cells
in the sample.
● The growth curve is NOT a
representation of a single cell’s
life cycle.

GC: Lag Phase

● Organisms metabolically
active, growing in size,
synthesising enzymes, producing energy in the form of ATP.
● Lag time depends upon the time microorganism takes to adapt to environment.
● Characteristics of media and organism influence lag time.

GC: Log Phase

● Exponential rate of growth.
● Cell division at the most rapid rate.
● Cells are dividing regularly by binary fission.
● Generation time (doubling time); the time it takes for the population to double.
● Generation time (G) is defined as the time (t) per generation (n = number of
generations). – G = t / n
● The growth of the organism is affected by both physical and nutritional factors
GC: Stationary Phase
● Cell division decreases. Number of new cells = number of old cells dying.
● Most natural environments produce stationary phase growth

GC: Death Phase

● Number of live cells decreasing

Essentials for Growth

● All organisms require three basic things for growth:
- A source of energy
- A source electrons for cellular respiration
- A source of matter for building additional cells: Carbon
● Microorganisms can be grouped into nutritional classes based on how they
satisfy their energy, electron and carbon requirements.

Energy Sources
● Phototrophs
- photo = self, trophe = nutrition.
- Carry out photon capture to acquire energy.
- Produces complex organic compounds from simple inorganic molecules
using energy from light.
- Other autotrophs may use other inorganic chemical reactions
● Chemotrophs
- chemo = chemical, trophe = nutrition.
- Obtain energy by the oxidation of electron donors.
- Donor molecules can be organic (chemoorganotrophs) or inorganic
(chemolithotrophs)

Electron Sources
● Lithotrophs
- Inorganic substances used as the electron source
- Inorganic molecules are substances that don’t have carbon hydrogen
(C-H) bonds; generally simple and are not normally found in living things.
- Examples of inorganic electron sources are minerals, metals and salts
● Organotrophs
- Organic substances used as the electron source
- Organic molecules, substances that contain carbon hydrogen bonds, are
found in living things.
- The major classes of organic molecules include carbohydrates, proteins,
lipids and nucleic acids.
Carbon Requirements
● Autotrophs
- CO2 used as the sole or principal carbon source.
● Heterotrophs
- Reduced organic molecules as carbon source, eg. glucose

Major Nutritional Types

● Photolithotrophic autotrophs
- Energy Source – Light
- Electron source – Inorganic eg. H2O, H2, H2S
- Carbon source – CO2
- Examples: Purple & green sulfur bacteria Cyanobacteria
● Photoorganotrophic heterotrophs
- Energy Source – Light
- Electron source – Organic
- Carbon source – Organic carbon (CO2 may be used)
- Examples: Purple non-sulfur bacteria Green non-sulfur bacteria
● Chemolithotrophic autotrophs
- Energy Source – Chemical (inorganic)
- Electron source – Inorganic
- Carbon source – CO2
- Examples: Sulfur-oxidising bacteria Hydrogen bacteria Nitrifying bacteria
Iron-oxidising bacteria
● Chemoorganotrophic heterotrophs
- Energy Source – Chemical (organic)
- Electron source – Organic
- Carbon source – Organic carbon
- Examples: Most non-photosynthetic bacteria
Lecture 5: Microbial Diversity and Novel Methods of Cell Culture
Microbiomes = Microbial Diversity
● Microbes make up the majority of the world's biomass!!
● All environments have their own microbiome
● >800 000 insect species exist each with their own specific bacterial communities
● Therefore, the number of species in the environment will continue to increase, by
orders of magnitude

Microbial Diversity Info is Increasing

● Molecular Revolution;
- 1983 PCR invented
- Primarily 16S rDNA sequencing
● Metagenomics
- shotgun, amplicon sequencing
● 92 phyla currently identified
● 5,000 Bacterial species have been isolated in the laboratory, characterised and
thus named

Traditional Cultivation
● Estimates predict up to 10^11 species
● Over 99.9% of bacteria are “unculturable” or recalcitrant to cultivation in the
laboratory into standard artificial media

Limitations of Trad. Cultivation

● Developed over a century ago
● Uses high concentrations of nutrients
● Biased towards fast growing species (r-strategists)
● Turbidity and colony development were measures of success at ~24 hrs

Candidate Phyla
● Entire Phyla known from 16S rDNA sequencing (Bacteria, Archaea and Fungi)
● Why can’t we culture them?
- We know little about their metabolic needs
- Substrates can be toxic
- Often rare, slow growing bacteria
- Require domestication

Microbial Dark Matter

● Environments surveyed are highly diverse: Soil, Biofilms, Seawater, Wastewater,
Sediments, Insects and Humans.
New Approaches to Culture ’non‐culturable’ Bacteria
● Extended incubation times
● Artificial media with limited nutrients
● Simulate the natural environment of the organism
● Isolation methods

Soil Substrate Membrane System (SSMS)

● A porous membrane serves as a barrier between the soil and the inoculum
● Soil is the media
● Growth as microcolonies requires microscopic visualisation
● Results in up to 60% of bacteria growing as microcolonies, including members
from the novel phyla TM7

Up to 60% Bacteria Now Recoverable

● Abundant bacterial growth
● Artificial media is toxic to many species
● Slow growing oligophilic bacteria need time
● Majority of growth is as microcolonies
● Traditional signs of growth would overlook the majority of growth
● But, many species still need to be domesticated
- That is to grow fast enough on artificial media to form a visible colony

How do we Domesticate a Novel Species?

● Once a microbe is enriched, pure colonies need to be obtained
● Slow growing species are usually K-strategists, they have a slow growing lifestyle
● In contrast, fast growing species like E. coli are R-strategists
● Domestication requires a switch to the R- lifestyle
- When subcultured repeatedly (>3 times), slow growers can be trained to
grow fast on artificial media
● Not always successful as we usually don’t know what the metabolic needs of
unknown bacteria are
Lecture 7: Industrial Enzymes
Macromolecules
● Macromolecules are chain-like molecules called polymers (except some lipids).
● Polymers are formed by covalent linkages of smaller units called monomers.

Polymer Monomer

Proteins Amino acids

Carbohydrates Monosaccharides

Lipids Fatty acids, glycerol

Nucleic Acid Nucleotides

Condensation of Polymers
● Macromolecules are made from smaller monomers in a dehydration synthesis
reaction in which an (-OH) from one monomer is linked to an (-H) from another
monomer.
● The reverse reaction, in which polymers are broken down into monomers, is
called a hydrolysis reaction.

Enzymes
● Enzymes are biological catalysts or catalytic proteins
● A catalyst is a chemical agent that changes the rate of a reaction without being
consumed by the reaction.
● Enzymes are highly specific:
- with respect to the reactions they catalyse.
- with respect to the choice of reactants (substrates).
● Enzymes have great catalytic power (can increase rates of reaction by > x10^6)
● Enzymes need certain surface shapes in order to bind substrates and release
products correctly

Activation Energy
● Chemical reactions involve the breaking and formation of bonds.
● The initial amount of energy required to start a chemical reaction is called the
activation energy, EA.
● This energy is often supplied in the form of thermal energy that the reactants
absorb from their surroundings.
 ● Enzymes speed up the reaction by lowering the activation energy, EA.
● They DO NOT affect equilibrium or the change in free energy, ΔG

Enzyme Structure
● Ionic bonds occur between
charged R groups (the side chain
of amino acids).
● Hydrogen bonds occur between
polar R groups.
● Hydrophobic interactions occur
between nonpolar R groups.
● Strong covalent bonds called
disulfide bridges can form
between the sulfhydryl groups
(SH) of two cysteine monomers
which act to hold parts of the
protein together
● Denaturation is the loss of a
protein’s normal three-dimensional
structure
● The rate of reaction is directly proportional to the enzyme concentration

Enzymes in Industry
● Enzymes are used throughout industry
● Required for most complex chemical conversions
● Massive worldwide investment in discovery and development of novel
bioprocesses
● The global industrial enzymes market should reach $7.0 billion by 2023.

Applications of Industrial Enzymes

● Food and dairy industry
- Amylase, protease, rennin, lipase, lactase
● Brewing industry
- Amylase, glucanases, protease
● Paper industry
- Amylase, cellulase, ligninase
● Biofuel industry
- Amylases, cellulases, ligninase
● Textiles industry
- Amylases, cellulases Bioremediation
Sources of Industrial Enzymes
● Microorganisms that can survive in extreme environments require enzymes that
can function under extreme conditions:
- Extreme heat
- Extreme cold
- High salt
- Acidic/alkaline environments
- High pressure

Bioethanol Production in Australia

● Approximately 500 million litres of bioethanol are produced annually in Australia
● Most of the ethanol used to make E10 in NSW is made by fermenting starch left
over after wheat has been turned into flour.
● The starch is fermented and converted into ethanol

Why Use Plants?

● Plants primarily store energy from sunlight in the form of starch molecules.

Amylase
● In Humans:
- Produced in the salivary glands and by the pancreas.
- Digests starch (large molecules) into simple sugars (smaller molecules)
that can be transported into the body and used to generate energy (ATP).
- Similar to other hydrolytic enzymes that break down proteins and fats.
● In Bacteria:
- Digests starch (large molecules) into simple sugars (smaller molecules)
that can be transported into the cell and used to generate energy (ATP).
Lecture 9: Growing Cells On A Large-scale To Produce Useful
Products
Examples of cell culture on a large-scale
● Brewing and Wine making
● Wastewater treatment
● Enzyme production by bacteria and yeasts
● Baker’s yeast production
● Ethanol production
● Oils and nutraceuticals from algae
● Amino acid production
● Production of vaccines and therapeutic proteins

Examples of Cells and Products

Cell Type Product

Bacteria Amino acids, enzymes, rProteins, pDNA

Fungi Secondary Metabolites

Algae Polysaccharides, oils

Insect Cells rProteins, virus, VLPs

Mammalian Cells rProteins, virus

Yeast Biomass, enzymes, rProteins, Ethanol,

beverages

Requirements for bioreactors for large-scale cell cultivation

● Fit-for-purpose
- Cell type, scale, materials, GMP?
● Controlled bioreactor environment
- Temperature
- Oxygen (level or absence?)
- pH
- Feed rate in fed-cultures
- Photons for phototrophs
● Can be cleaned and sterilised
- Or disposed of?
● Sterility can be maintained
Cell Type
● Size, morphology and fragility
● Requirements for surface growth
● Aerobic or anaerobic
- Oxygen demand can be high for large-scale, high density fermentations
● Temperature requirements
● Cell physiology
- eg. Anchorage dependence

Continuous Culture
● As with chemostats in chemical reactions, the continuous bioreactor can come to
a steady state.
● F1 = F2 = F
● Growth rate can be controlled and equals the dilution rate (F/V) at steady state

Bioreactor Designs
● Materials
- Plastic, glass, steel, stainless steel, concrete
● Scale
- Millilitres to > 106 L
● Extent of instrumentation and control
- Temperature, oxygen, pH, other
● Cleaning and sanitation in-place
● Examples
- Stirred tanks, column and tower fermenters, bags and bottles, raceways

Bioreactor requirements depend on the product or process

● Requirements for:
- Cleaning, sanitisation, sterilisation
● End-use of product
- Industrial, food/beverage, therapeutic
● Validation requirements
- Food and therapeutics industries
● Scale of production

Photobioreactors
● Culture of photosynthetic organisms to produce
- Oils for biodiesel
- Valuable nutraceuticals
- Other fine chemicals
 ● Microalgae, cyanobacteria
● Carbon capture

Closed Systems vs Open Ponds

● Advantages of closed systems:
- Possible to avoid infection by unwanted strains (e.g. having lower oil
content)
- Possible to avoid infestation by bugs that eat algae
- Very good control of conditions (light, nutrients, CO2 levels) = growth rate
and/or oil yield can be optimised.
- CO2 capture facilitated
- Water conservation
● Advantages of open systems:
- Much, much cheaper in terms of capital cost

Summary
● Bioreactors vary in:
- Size
- Type and cost of materials used
- Instrumentation and control requirements
- Validation Requirements
● Single-use bioreactors are becoming favoured in pharmaceutical applications
● Photobioreactor systems for “green chemistry” applications
Lecture 10: Beer Brewing
Beer Ingredients
● Water
● Hops
- Flavour (bitterness), aroma
● Malt
- sugars, proteins, colour, flavour
● Adjunct
- extra fermentable sugars
● Yeast
- top and bottom fermenting strains of Saccharomyces
- converts sugar to ethanol and produces flavour compounds

Beer Attributes
● Appearance
- Foamy “head”
- Amber to dark colour
- Cloudy or clear
● Alcohol 1-12% (v/v) (typically 4-6%)
● Taste and Aroma
- Bitterness from the hops
- Floral notes from hops and minor alcohols
- Carbonation: acidic “bite”
- Alcohol

Global Beer Production

● Global beer production amounted to 187 million kiloliters in 2016
● Asia (down 1.6% year-on-year with a share of 33.9%) marked its 6th consecutive
year as the top producing region. Africa (up 2.6% year on-year with a share of
7.3%), marked its 6th consecutive year of growth.
● China remains the largest beer producing country for 14 consecutive years,
followed by the United States.
● Australia ranks 24th

Biotechnology in Brewing
● Enzyme catalysed “hydrolysis”
- Starches to Glucose
- Proteins to peptides
● Protein denaturation
 - Boiling of wort denatures proteins, causing them to become insoluble and
capable of removal from the wort, improving beer clarity and stability
● Yeast cell growth in fermenters on malt sugars in the fermentation
- Cell growth, ethanol production

Constraints on the Process

● Yeast
- optimum growth around pH 5, 30-35 oC, ethanol tolerant, to
- Brewery fermentations typically at lower temperatures: 12- 20 oC
● Contaminants
- Wild yeasts, lactic acid bacteria
● Oxygen (should be excluded, except @ fermentation start)
● Wort preparation
- ingredients
- wort production method
● Somewhat unstable
- light and oxygen sensitive
- may become contaminated
- However it is low pH, 3-10% v/v ethanol and compounds in hop extracts
are antibacterial
● Carbonated
● Beverage: consumer products
- sensitive market
The Overall Process
Water Treatment
● modify hardness
● remove organics and suspended solids
● sterilise
● carbonate/de-aerate for adjusting and sparging waters

Malt
● Malt is selected to give desired flavour, aroma and colour
● Milled to break husk and allow access to grain contents
● Qualities affected by starting material and temperatures used
- Higher temperature and longer kilning result in darker malts with lower
enzyme activity
● Malt is modified barley or wheat: objective is to partly modify the grain by
beginning seed germination, then terminating germination by heating.
● A key feature of malting grain is to generate enzymes to make the grain
fermentable:
- Starches become Maltose through amylase
- Proteins become Smaller Peptides through proteases
● Steps in malting are: steeping, germination and kilning
● Process:
- Steeping - raises water content of grain to begin seedling formation
- Germination - grain sprouts forming plant embryo: important enzymes
develop
- Kilning - heat: growth arrest, colour and flavour development

Mashing Process
● Malt and adjuncts suspended in water
● Starches gelatinized and then converted to sugars
● temperature profile important for enzyme action to work
● mash filtered in mash filter or lauter tun to separate solids from “sweet wort“
- Typically re-sparged to ensure good recovery of sugars

Enzymes
● Biological protein catalysts from malted grain
- Enzyme generation in the malting process is very important
● Alpha and Beta-amylases to produce glucose, maltose and dextrins from
starches
● proteases to produce smaller peptides and amino nitrogen for yeast nutrition
● Amylases:
- Breaks down starches into smaller sugars
 - Those which the yeast can use are termed "fermentable“
- Yeasts cannot utilise starch; grain must be malted to be fermentable
● Proteases:
- Break proteins down into smaller peptides
- Gives the yeast nutrients to grow on affects physical and "mouthfeel"
properties of beer

Filtering/Lautering
● separate the extract (basis for wort) from the solids (grain husks from malt)
● mash filters or more commonly, “Lauter tuns”
● grain husks form filter bed
● sparging to increase extract yield
Brew Kettle and Whirlpool
● Kettle sterilises wort and releases/modifies hop flavour and aroma compounds
- Hops added periodically as “gifts”
● proteins denatured (they come out of solution)
● Whirlpool used to clarify wort from “trub” Trub is denatured protein, any
remaining grain husks and hop flowers (if added)
Post Brewing and Fermentation
● Yeast settling
● Filtration of clarified beer
● Pasteurisation
● Packaging
● Beer product is characterised by:
- Appearance
- Flavour
- Alcohol level
- Aroma
- The “head”
Summary of Brewing Operations
● Beer quality is critically determined by wort and yeast preparation
● Water, malt, adjuncts, hops are treated and added at different stages
● Yeast is freshly prepared or recycled
● Pitching denotes the start of the fermentation - oxygen required here for the
yeast to get started
● Operations post-fermentation include filtration, pasteurisation and packaging
● Key bio-reactions are:
- The enzymic conversion of starches and proteins by malt enzymes
- The fermentation of wort sugars to produce ethanol, yeast, carbon dioxide
and flavor compound
Lecture 12: Genetics Review
● DNA = Deoxyribonucleic acid
● RNA = Ribonucleic acid
● A always pairs with T
● C always pairs with G

Gene Expression
● DNA contains the instructions for how a cell will function
● Proteins perform these functions
● How are the instructions translated from the nucleotide language of DNA into the
amino acid language of proteins? Via an intermediary - RNA

Transcription
● Rna is created, where T is replaced with Uracil

Translation
● RNA -> protein
● The protein synthesis factory is called the ribosome
● tRNAs carrying their amino acids move to the ribosome
● They base-pair with the mRNA
● This places their amino acids in the correct sequence to form the protein that was
coded in the mRNA (and hence in the DNA sequence)

How is the mRNA translated into amino acids? There is a “go between: t-RNA

t-RNA
● There is a separate tRNA for every amino acid
● The amino acid is attached to one end of the tRNA
● The other end has a triplet code that will base-pair with the mRNA

Control of Gene Expression

● In prokaryotes:
- Control of gene expression occurs at the level of transcription, i.e. whether
a gene is transcribed or not.
- Once the mRNA is formed it is translated immediately
● In eukaryotes:
- The mRNA is formed as pre-mRNA in the nucleus (where the DNA is!)
- The pre-mRNA is extensively modified
- It is then exported to the cytoplasm, where protein synthesis occurs
- The control of gene expression occurs at many levels.
 - Other levels include the processing, transport and degradation of the
mRNA.
- At the protein level, proteins can be modified, transported and degraded.

Initiation of Transcription in Eukaryotes

● Proteins called transcription factors mediate the initiation of transcription
● This is an additional control mechanism for gene expression in eukaryotes
● Activators are also required for eukaryotic gene transcription
Lecture 13: Genetic Modification
Definition
● Genetic modification n. alteration of genes, esp.by selective breeding or (in later
use) genetic engineering; an instance of this
● Genetic modification (GM) is the use of modern biotechnology techniques to
change the genes of an organism, such as a plant or animal

Conventional Genetics
● Evolution/natural selection
● Mutations
● Selective breeding

Mutations
● Changes in the nucleotide sequence of DNA
● Major inducer of genetic diversity (evolution)
● Can produce useful or detrimental changes
● Causative agents : “Natural” errors, mutagens, UV radiation
● Single nucleotide mutations (most common) change the DNA triplet
- results in change in mRNA codon
- may lead to changes in the amino acid sequence of the encoded
polypeptide
- can lead to disease

Base Substitution Mutations

● Missense Mutations - Changes one sense codon to one that specifies a different
amino acid
● Nonsense Mutations - Changes a sense codon to a stop codon
● Silent Mutations - Changes one sense codon to another sense codon that
specifies the same amino acid

Frameshift Mutations
● Base-pair insertion or deletion - alters the reading frame after the point of the
mutation

Inherited Mutation - Passed on through gametes (sperm or egg cells)

● All cells of organism will have the same mutation
● Birth defects and inherited disease

Acquired Mutation - Occur in the genome of somatic cells

● Not passed onto future generations
 ● Can give rise to abnormalities in cell growth, e.g. cancers

Genotype - The entirety of an organism’s genetic material

Phenotype - The set of observable/physical traits of an organism

Conventional Genetic Techniques

● Any manipulation used to create or select for altered phenotype(s) in an
organism or cell line without directed recombination of DNA
● Eg:
- Crossing strains of wheat to get ‘rust resistant‛ wheat (W.J. Farrar 1880‛s)
- Crossing strains of merino sheep to get better wool yields (Governor
Macarthur, 1800‛s)

Recombinant DNA Technology and Genetic Engineering

● Genetic engineering uses recombinant DNA technology to alter genes for
practical purposes
● Recombinant DNA contains DNA from multiple sources joined together
- Recombinant vectors containing the gene of interest can be cloned in E.
coli
- Recombinant DNA technology: isolate, purify, analyze and manipulate
DNA sequences
- DNA fingerprinting used in forensics
- Microarray for studying gene expression
Lecture 14: Recombinant Technology
● Genetic engineering uses recombinant DNA technology to alter genes for
practical purposes
● Recombinant DNA contains DNA from multiple sources joined together
- Recombinant vectors containing the gene of interest can be cloned in E.
coli
● Recombinant DNA technology: isolate, purify, analyze and manipulate DNA
sequences

Overview

Overview of Gene Cloning

Techniques
Clever Gene Manipulation Techniques
● Cutting DNA into pieces – restriction enzymes
● Pasting DNA pieces together – ligase
● Cloning your special piece of DNA – vectors
● Expressing your gene – hosts
● Nearly every one of these techniques exploits a natural phenomenon developed
by nature

Restriction Enzymes
● Restriction enzymes (RE) are endonucleases that are isolated from bacteria
● Found because they restrict the growth of infecting viruses
● Restriction enzymes can be used to cut DNA from different sources.
● If they produce sticky ends, these DNA fragments can be mixed and joined

Ligase

Vectors
Once you have isolated your gene, you need to place it in a vector so that you can:
● Preserve it
● Make lots of copies of it
● Manipulate it
● Express it
Plasmid Vectors
● Plasmids – circular extrachromosomal DNA in bacteria
● Bacteria use plasmids to transfer traits (like antibiotic resistance) from one to the
other
● We can insert DNA into the plasmids – the bacteria will replicate the plasmid

Hosts
● The plasmid containing the gene needs to be placed in a host cell that will
replicate the plasmid
● Two techniques are commonly used:
- CaCl2 transformation
- Electroporation

CaCl2 transformation
● Bacteria and DNA mixed with CaCl2
● Ca2+ ions help to neutralize negative charges on DNA and cell membrane
● Lower temperature slows movement of phospholipids – easier to shield with
Ca2+ ions
● Then raise temperature (“heat shock”) so DNA can move through membrane into
cell

Lecture 15: Recombinant Technology 2

cDNA
● Complementary DNA (cDNA) can be made from mRNA (expressed sequences)
● Using cDNA, the coding sequence for a human gene can be cloned and
expressed in bacteria

DNA Microarray
● DNA microarray assay for gene expression
● The expression levels of thousands of genes can be determined on one array
● Using bioinformatic tools can compare gene expression levels between normal
and cancer

PCR - Polymerase Chain Reaction

● During PCR, the Taq polymerase adds an extra A on the 3’ end of the product
● We can exploit this to make it easy to insert the PCR product into a vector that
has T overhangs
Commercial Vector - pGEM
The virtues of this vector are that:
● PCR products can be readily inserted
● The insert can be sequenced using T7 or SP6 primers
● The insert can be easily removed using the flanking RE sites
Genes are inserted into expression vectors so that they have a promoter - allows RNA
polymerase to transcribe the gene

Transgenic Plants
Genetically engineered plants have genes inserted that:
● Provide resistance to insects
● Provide resistance to herbicides
● Fix nitrogen (reduces need for chemical fertilizers)
● Produce vitamins and human proteins
● Produce viral proteins for use as vaccines

Transgenic Animals
● Transgenic mice used to investigate gene function and as models for human
gene therapy
- To produce transgenic mice cloned DNA is microinjected into a fertilized
egg
● Large animals created for the production of better livestock and for synthesizing
human proteins in milk (>10g/l)

“Pharm” Animals
● There are specially bred sheep which express a human protein in their milk
● The protein is readily purified
● Being tested as a treatment for cystic fibrosis
Lecture 16: Commercialisation of Scientific Discoveries
Areas of Commercialisation
● Medicine
● Veterinary
● Agriculture
● Environment
● Forensic
● Manufacture
● Food
● Enabling technology (tools)

Medicine
● Preventing and treating disease
- Vaccines
- Cancer
- Aging
- Autoimmune disease
- Neurodegenerative diseases

Agriculture
● Improving quality and quantity

Environment
● Contamination
● Water treatment
● Algal blooms
● Climate change
● No (coal)
● Yes (nuclear)

Forensic
● Individual identification at crime scenes
● Yesterday: fingerprints
● Today: DNA, mass spectrometry
● Tomorrow: ?

Manufacturing
● Products:
- ethanol and other valuable biochemicals
- antibodies
 - vaccines
- enzymes
- hormones
- sugars
- proteins
- RNA/DNA
- Antibiotics
● Methods:
- microbial fermentation
- tissue culture
- chemical synthesis
- biocatalysis
- wild or transgenic plants & animals
- extraction from human blood and other tissue (cadaver)

Food
● Microbial foods:
- Bakers yeast (bread, alcohol)
- Lactic acid bacteria (LAB) = cheese, yoghurt, salami
● Problems to overcome:
- Phage infection (LAB)
- Biomass yields and productivity
- Activity
- Flavour profiles
- Stability

Enabling Technologies
● Recombinant DNA
● Gene editing (e.g. CRISPR)
● DNA sequencing
● Mass spectrometry (e.g. proteomics)
● Stem cells
● Machinery & Equipment

Types of Research
● Fundamental (basic research)
- Curiosity driven
● Applied (translational research)
- Market driven (designed to address an unmet need) o e.g. need for
electric vehicles
 - Technology driven (find a use for a discovery) o e.g. CRISPR gene editing
was found by curiosity, but its discovery enabled us to apply CRISPR for
gene therapy
- Empirical (discover something that solves a problem, then work out how it
works), e.g. drug screen to find a molecule that kills cancer cells, then
figure out how the molecule works, then make more drugs that work the
same way.

Patent
● A patent gives the holder the right to exclude others from making, using, selling,
offering to sell, and importing any patented invention
● Allows a monopoly for a certain period of time by keeping the competition out.
● Without patents, it is hard to commercialise a technology
● The minimum requirements for obtaining a patent are that the invention must be:
- novel
- non-obvious or include an inventive step
- useful or capable of commercial application

Common Ways to Get Funding

● Federal government (does not take equity)
● Industry (may take equity)
● Venture capital groups (will take equity)
● University seed funding (usually will take equity)
Lecture 17: Vaccines
Definition of Vaccine
● Vaccines can be defined as a product that stimulates a person’s immune system
to produce immunity to a specific disease, protecting the person from that
disease.
OR……
● A vaccine can be defined as a substance used to stimulate the production of
antibodies and provide immunity against one or several diseases, prepared from
the causative agent of a disease, its products, or a synthetic substitute, treated to
act as an antigen without inducing the disease

Viruses
● A virus is a particle composed of proteins and nucleic acid, much smaller than a
cell, and does not fulfill all the required characteristics of a living cell, the smallest
form of life. It is NOT alive.
● It does not’ ‘grow’ or become larger, and it does not take up nutrients and
produce waste.
● However, it does reproduce, but with the assistance of its host cell machinery.
● The viral nucleic acid can be either DNA or RNA, so depending on the type of
nucleic acid in the virus, we can consider viruses as either DNA or RNA viruses
● Viral nucleic acid genomes can also be linear single stranded nucleic acids or
linear double stranded; or the viral genomic can be circular, in either single or
double stranded forms

How do vaccines use our Immune systems to prevent, reduce, or completely

block the effects of viral infection?
● Your immune system is composed of the INNATE immune system and the
ADAPTIVE immune system.
● Innate Immune System
- The innate system is already active within you, before you ‘see’ a foreign
protein or virus or bacteria or PATHOGENS.
- Specialised cells such as macrophage and neutrophils and dendritic cells
will be able to defend against infection, being attracted to the infection site
and literally ingesting the infected cells and pathogen.
- The innate immune response is very quick….in fact, the response to an
infection on your skin can be as quick as a couple of hours.
● Adaptive Immune System
- It also requires those macrophage or dendritic cells, but takes much
longer than the innate immune system to mount a response because the
 adaptive immune response also relies on specific B and T cells which
need to be activated to proliferate to large numbers.
- Cells such as the macrophage and B cells, will ingest the pathogen,
degrading the pathogen proteins into small peptides, and then these
peptides will associate with the MHC molecule on the surface of the
dendritic cell
- A T cell receptor, expressed on the surface of a T cell, recognizes and
binds to the pathogen peptide/MHC complex, resulting in activation of the
T cell as well as activation of specific B cells
- B cell activation is critical for production of specific and secreted
antibodies as well as for generating memory B cells
- Activation of both specific T and B cells leads to rapid proliferation of these
specific cells during the first PRIMING event.
- Then subsequent infection allows these cells, including memory B and T
cells, to remember the infection and fight it effectively.
- In summary, ADAPTIVE IMMUNITY results from the specific activation of
B cells and T cells so that there are MEMORY B cells and Cytotoxic T
cells to fight future infections!
- Activation of the immune system is HIGHLY specific…..but not perfect!
And some of the side effects we will consider with vaccination are due to
activation of the immune system.

Vaccine Mechanisms of Action

There are 4 traditional types of vaccines:
● Traditional ‘whole’ vaccines
- Stimulate the production of antibodies via challenges with purified proteins
from the pathogens, or by using whole cells (live, attenuated vaccines).
● Protein subunit vaccines
- They use a piece or protein of the virus, such as the S protein from the
SARS CoV2 virus
● Harmless Viral vector vaccines
- Enables expression of viral nucleic acid and viral proteins.
● Nucleic acid DNA or RNA vaccines
- They use RNA or DNA that upon entering cells, are translated to antigenic
molecules that in turn, stimulate the immune system. The production of
RNA-based vaccines is more rapid and less expensive than traditional
vaccines. Messenger RNA has the advantage of immediate protein
production in the cytosol and reduced concerns over genomic integration.
‘Whole’ Virus Vaccines
● TWO main approaches:
- Live attenuated vaccines use a weakened form of the virus that can still
replicate without causing illness.
- Inactivated vaccines use viruses whose genetic material has been
destroyed so they cannot replicate, but can still trigger an immune
response.
● Whole virus vaccines are reproduced by growing the virus in large amounts,
isolating it and inactivating it. For influenza or flu vaccines, the virus is grown in
fertilized chicken eggs, and for other viruses can be grown in cells in culture

Protein Subunit Vaccines

● Subunit vaccines use pieces of the pathogen - often fragments of protein - to
trigger an immune response.
- Minimises the risk of side effects, but it also means the immune response
may be weaker.
- Often require adjuvants, to help boost the immune response.
- An example of an existing subunit vaccine is the hepatitis B vaccine
● The protein subunits are produced recombinantly, being expressed in yeast cells,
and then protein subunits isolated, so this can be an expensive process and
maintaining stability of the often large synthetic proteins can be a challenge

Viral Vector Vaccines

● Involves inserting the nucleic acid corresponding to a viral protein antigen into
the vector, which gets into the host cell and uses the host’ cells own machinery to
translate the viral antigenic protein for expression on the cell surface.
● There are two main types of viral vector-based vaccines:
- Non-replicating vector vaccines are unable to make new viral particles;
they only produce the vaccine antigen.
- Replicating vector vaccines also produce new viral particles in the cells
they infect, which then go on to infect new cells that will also make the
vaccine antigen.

Nucleic Acid Vaccines

● Involves producing the DNA or RNA which encodes an antigenic viral protein,
and then introducing that nucleic acid into cells.
● While there are DNA vaccine sin development, these require that the DNA be
inserted into a plasmid
● On the other hand RNA vaccines encode the antigen of interest in messenger
RNA (mRNA) or self-amplifying RNA (saRNA)
 ● Because RNA is just a temporary copy of the DNA, there is little risk of it
integrating with our own genetic material. The RNA can be injected by itself or
can be protected or encapsulated within lipid nanoparticles
● Once the DNA or RNA is inside the cell and it starts producing antigens, these
are then displayed on its surface, where they can be detected by the immune
system, triggering a response
- Includes killer T cells, which seek out and destroy infected cells, as well as
antibody-producing B cells and helper T cells which support antibody
production

Herd Immunity
● The idea that when ENOUGH people in the community are vaccinated or
immune, then the spread of the disease is contained.
● To prevent transmission of the highly infectious measles, we need 95%
vaccinated for containment. And we have eradicated smallpox.

Variants
● As viruses infect many people, causing many infections, there are more chances
for the virus to mutate
● mutating simply means that the virus makes mistakes in copying its RNA
● introducing changes in the nucleic acid which result in changes in the proteins,
such as in the S proteins
● Many mutations occur ,some disappear, some are retained. But those that
provide an advantage to the viral growth and transmission, are those that we are
most concerned about.
Lecture 18: Medical Mycology and Biotechnology
Definition
● Medical biotechnology is the use of living cells and cell materials to research and
produce pharmaceutical and diagnostic products that help treat and prevent
human diseases.

Mycology: The Study of Fungi

● The word mycology comes from the Greek: μύκης (mukēs), meaning "fungus"
and the suffix -λογία (- logia), meaning "study"
● A fungus is:
- A chemo-organotrophic eukaryote that lacks chlorophyll and forms spores
- its cell wall contains polysaccharides, often chitin or cellulose, and it
absorbs nutrients
- its membrane contains ergosterol as the major sterol
- Classification is principally based on morphology
- Mushrooms, moulds, yeast
● Fungi can infect plants, insects, amphibians and mammals

Fungal Infections of Humans

Superficial (“on”)
● Diseases caused by the dermatophytes
- Ringworm
- Tinea
- Athlete’s foot
● Diseases caused by Candida spp.
- Thrush, in mouth, vagina, penis, nails and skin
These affect 25% of the world's population!

Invasive/Systemic (“in”)
● Candidiasis
● Aspergillosis
● Cryptococcosis
● Mucormycosis
● Pneumocystis

Opportunistic Invasive Fungal Pathogens

● 2 million life-threatening infections per year world wide
● Mortality: 20 – 95%
● More people die from invasive fungal diseases than from tuberculosis or malaria
Candida Albicans and Disease
● Most common serious fungal pathogen of humans
- >400,000 life-threatening infections per year world wide (~350/year in
Australia)
● The GI tract of at least 50% of humans is colonised with C. albicans without
apparent ill effect
● Groups at risk from invasive Candida infections include:
- Cancer patients
- organ and bone marrow transplant recipients
- Those receiving immunosuppressive therapy
- patients with AIDS
- major surgery
- ICU
● 46-75% mortality, even with antifungal treatment
● Culture is still the gold standard for diagnosis
● Inadequate range of antifungal drugs
● Patient outcome is improved with rapid diagnosis and treatment

Current diagnostics - candidiasis

● Blood culture
- 50% sensitivity (misses half of the positives)
- ID to species level = 72 h
● Molecular-based diagnostics (e.g. PCR)
- DNA extraction issues
- Cannot differentiate between colonisation and systemic disease

Antibiotics Vs Antifungals
● Antibiotic
- any substance that inhibits the growth and replication of a bacterium or
kills it outright
- The term “antibiotic” refers only to an antibacterial agent
- Antibiotics work by affecting things that bacterial cells have that human
cells do not

Fungi are eukaryotes. How will we kill them without killing ourselves?
● A fungus is:
- a chemo-organotrophic eukaryote that lacks chlorophyll and forms spores
- its cell wall contains polysaccharides, often chitin or cellulose, and it
absorbs nutrients
- its membrane contains ergosterol as the major sterol
 ● Potential targets for antifungals are:
- ergosterol synthesis (endoplasmic reticulum)
- Cell wall
- Protein synthesis
- cell membrane
- nucleus (DNA/RNA synthesis)

Classes of Antifungal Drugs

● Echinocandins
- e.g. Caspofungin
● Polyenes
- e.g. Amphotericin B
● Azoles
- e.g. Fluconazole
Research areas in the Lenardon group
● Cell wall structure and biosynthesis
● Cell division and septation in Candida albicans
● Antibody-based therapies and diagnostics for fungal infections
● C. albicans colonisation of the colon

What are Antibodies?

● An antibody is a protein produced by the immune system (B cells) that is capable
of binding with high specificity to an antigen.
● These antigens are typically other proteins, but may be carbohydrates, small
molecules or even nucleotides.
● Antibodies only bind part of an antigen – and the specific part of an antigen to
which they bind is called an epitope.
● The specificity of binding of antibodies to their epitope can be exploited to detect
a specific target in an assay.
● Antigen binding is via the CDRs (complementarity determining regions).
● The DNA sequence encoding the CDRs determines specificity

Polyclonal and Monoclonal Abs

● Immunisation of an animal against an antigen results in MULTIPLE antibodies
with different affinities to the same or different epitopes on the antigen.
● Sera from such an immunised animal is termed polyclonal.
● The DNA sequences encoding the CDRs of each antibody in the preparation will
be different.
● Monoclonal antibodies (mAbs) are produced from single cells in a laboratory
environment.
 ● Each mAb binds ONE unique epitope.
● The DNA sequence encoding the CDRs of the mAbs in a preparation will be the
same

Antibody Fragments

mAbs as an alternative to antifungal drugs

● Problems with antifungal drugs
- No new small-molecule antifungal drugs have been approved in over 15
years
- Because humans and fungi are both eukaryotes, there are very few
differences between fungal cells and human cells that can be exploited
- Antifungal drugs can have toxic side effects if subtle differences are
exploited, e.g. ergosterol and cholesterol
- Antifungal resistance is on the rise
● Properties of mABS
- mAbs recognise a single epitope with high specificity
- even very small differences between fungal cells and human cells can be
easily exploited
- eliminates the possibility of off target and toxic side effects
- Human mAbs are not toxic to humans
Lecture 20: Global Impact
Microorganism’s Impacts on Global Issues
● Our evolutionary history and future
● Our nutritional survival - eg. N 2 fixation, food production
● Climate change - global nutrient cycling
● Pandemics - diseases and health
● New drug discovery
● Bioterrorism
● GMO
● Bioremediation - clean up of waste

Impact of biotic & abiotic factors

● All natural environments are impacted by biotic factors: interactions, grazing,
viruses, antibiotics
● All natural environments are impacted by abiotic factors: water, temperature,
light, oxygen, nutrients
● All natural environments are nutrient-limited (C, N, P, Fe)
● All nutrients in the environment are cycled
● The controlling factors (abiotic or biotic) can change over time
- natural environments are dynamic
- complex community compositional changes
● To understand
- base-line
- monitor

Genomics Revolution
● first bacterial genome – 1995
● First environmental genome (metagenome) – 2004
● Metagenomics: determine which genes are present in whole environmental
samples

Carbon Cycle
Iron Cycle

Microbial Processes
● Abiotic acid generation from pyrite
- 2FeS 2 + 2H 2O + 7O 2 = 2Fe 2^+ + 4SO 4 2^− + 4H^+(aq)
- Pyrite + water + oxygen = ferrous iron + sulfate + acid
● Microbial acidification
- Acidithiobacillus ferrooxidans lives in iron-sulfur minerals (pyrite)
- oxidises iron + sulfur as energy
- grows autotrophically (CO 2)
- produces ferric iron + sulfuric acid
- produces ferric ion catalyst (Fe 3 +) – accelerates pyritic oxidation
10^6-fold
- Fe 2^+ + ¼ O 2 + H^+ → Fe 3^+ + ½ H 2 O
● Microbial Syntrophy
- A. ferrooxidans better CO 2 uptake
- Sulfobacillus thermosulfidooxidans better Fe 2^+ oxidation
- Help each other to grow = faster Fe 2^+ oxidation (acidification)
The Microbiologists’ Warning
● A Consensus Statement proclaiming that microorganisms are so critical to
achieving an environmentally sustainable future that ignoring them risks the fate
of Humanity
● Microbes Impact Climate Change – Climate Change Impacts Microbes Marine...
Agriculture... Mitigation…
● Human beings and the natural world are on a collision course.
● Human activities – harsh – irreversible damage – environment – critical
resources
● Current practices put at serious risk the future t– human society – plant and
animal kingdoms – unable to sustain life
● Fundamental changes are urgent if we are to avoid the collision our present
course will bring about

Microbes Impact Climate Change

● CO 2 fixation & O 2 production
- marine = half net global
● GHG emissions
- heterotrophic respiration (CO 2 )
- methanogenesis (CH 4 )
- denitrification (N 2O)

Climate Change Impacts Microbes

● community composition
● functional processes
● physiological responses
● evolutionary adaptation
● Marine Impacts:
↓Sea ice = ↓habitat = ↓CO2 fixation = ↓microbes = starving system
- corals – warming, acidification, eutrophication, overuse
- microbiome changes; replaced by macroalgae & cyanobacterial mats
Higher CO2 = irreversible increase N2 fixation & growth rate – cyanobacteria
- Intracellular pH balance – impact nutrient cycling
- algae – cell size, growth rate, ecotype diversity
- algae – non-toxic → toxic
- viruses – diversity
Warming = algae changing ribosome concentrations (P-rich) → P:N ratio
- poleward shift in microbial populations
● Terrestrial Impacts:
Temperature, precipitation, soil type = differential impact/response
 - growth efficiency changes = ↑degradation recalcitrant material
- warmer = ↑microbial lifestyles Climate Change Impacts Microbes
- change microbial diversity/function = change plant diversity/survival
- evolutionary adaptation patterns unclear
Permafrost = carbon sinks opening → reservoirs = ↑microbial respiration = ↑emissions
- thawing → community change
- plant expansion = ↑root exudates = ↑microbial activity
Displacement of plant species = ↑microbial activity → ↑N (peptides)
- indigenous moss → Antarctic hair grass, better adapted to N
Antarctic temperature increase
- changed fungal and cyanobacterial community
- ↑toxic freshwater lake cyanobacteria
● Agriculture Impacts:
Crops
- extensive (grassland) = fungi = drought resistant
- intensive (wheat) = bacteria = less drought resistant
- ↑aridity = ↓fungal & bacterial diversity = ↓plant diversity
Eutrophication & warming
- changed microbial communities
- ↑toxic cyanobacterial blooms
- ↓thermal mixing
● Pathogen Impacts
Marine warming & acidification
- ↑coral pathogens
- ↑fish disease
- ↓starfish = ↑urchins = ↓kelp forests
↑Crop disease
- ↑forest die-off (plant stress)
- ↑tree frog disease (temperature fluctuation)
↑Pathogen spread
- environment – warming
- transport – human activity
Vector-borne, waterborne, airborne, foodborne
- ↑distribution vectors (mosquitos)
- ↑poleward spread waterborne (Vibrio)
- ↑rates pathogen replication
- ↑antibiotic resistance spread
Warming = ↑growth rates
- ↑environmental persistence
- ↑carriage
 - ↑transmission
- ↑HGT
- increase human population density
● Mitigation of Impacts:
- Wolbachia – mosquitoes = ↓transmission (dengue)
- ↑bacterial N2O → N2 = ↓soybean emissions
Ruminant breeding = ↓methanogens, CH4
- Microbial mycoprotein meat substitutes
- Bromoform (red seaweed) = ↓methanogens, CH4
- Biochar retain organic matter = ↓microbial remineralization
Constructed wet-lands
- cellulosic biofuel
- ~7% China gasoline
- wet-land emissions?
Microbial technologies = practical solutions
- chemicals, materials, energy, remediation
- United Nations Sustainable Development Goals: Poverty, hunger, health,
clean water, clean energy, economic growth, industry innovation,
sustainable cities, responsible consumption, climate action, life below
water, life on land
Lecture 21: Bioremediation
The problem: Xenobiotics
● Man Made compounds which are non-biodegradable and hence accumulate in
the environment
● Are man made chemicals found in organisms that are not expected to be
produced or present in them
● In some cases they are produced naturally at very low concentrations, but are
present in nature at very high concentrations.

Xenobiotic compounds
● Pollutants produced by industry like pesticides, polychlorinated biphenyls
(PCBs), Dioxins, dyes and chlorinated solvents
● Drugs- Antibiotics in humans
● Pesticides
- Common components of toxic wastes
- Include herbicides, insecticides, and fungicides
- Represent a wide variety of chemicals, some of which can be used as
carbon sources and electron donors by microbes

The Age of Remediation

● Resource conservation and Recovery Act 1980
● Emphasis on management of new waste and remediation of old sites
● Hazardous waste treatment/safe disposal
● Public awareness/stringent regulations/ environmental liability…..
● The focus is waste minimisation, resource recovery, recycling, on-site treatment,
detoxification of contaminants, remediation….
● Waste generators responsible for the fate of their waste

Cures
● Physical:
- Incineration
- Landfilling
- Dig and haul
- Soil washing
- Chemical treatments
- Solvent extraction
- Air sparging….
● Biological:
- Bioremediation
- Phytoremediation
What is Bioremediation?
● ‘Bioremediation is the process of cleaning up environmental sites contaminated
with chemical pollutants by using living organisms to degrade hazardous
materials into less toxic ones’

What is Biodegradation?
● ‘Biodegradation is the breakdown in materials caused by the direct and/or
indirect action of biological entities’

Advantages of Bioremediation
● Cost-effective
● In situ application
● Universal
● Destroy/detoxifies pollutants
● Removes liability
● Visually appealing

Three Major Bioremediation Strategies

● Natural Attenuation
● Biostimulation
● Bioaugmentation

In situ: Natural attenuation

● Takes time
● Depends on soil characteristics, nutrients, water…..
● Bioassessments are required to determine if the microbial population of interest
is present naturally at the site level…
● Requires site management
● Biodegradation usually occurs through the actions of a consortia of microbes…..
● Most cost-effective!

Biostimulation
● Site characterisation & biofeasability should be carried out to determine presence
of native indigenous microbial population containing specific
contaminant-degrading microbes
● If the natural population is present activity might need to be improved or
‘stimulated’
 ● A delay occurs as the microbial population assimilates, nutrients are not specific,
so all microbes potentially propagate
● These microbes will be able to biodegrade the target site contaminants IF
managed properly

Bioaugmentation
● Advantages: In situ-a specific population is injected into environment thus
degradation process starts immediately
● Efficiency relies on the selectivity and specialisation of microbes added
● But, the real potential is still debatable as the movement from lab-scale to the
field is never as promising
● "Microbes are a lot like teenagers, they are hard to control,” Chris Reddy of the
Woods Hole Oceanographic Institution.
● Using native environment has the advantage that microbes are more likely to
survive
● Indigenous organisms usually in low abundance
● Bioremediation relying on microbial consortia are more effective, like ‘real’
situations
- Richer metabolic network for exploitation
● Insecticides, chloroform and petroleum have been bioremediated from
groundwater and soil
● Major issue is selection and isolation of microbial consortia of interest-to link
function with the contaminant

Oil: a major contaminant

● Complex mixture of hydrocarbons, alkanes, aromatics and cycloalkanes
● Requires a consortia of microbes to break it down

Typical approaches to bioremediation of oil

● Dispersants are used to emulsify oil into small droplets, this is to aid the ability for
bacteria to degrade the material (mixtures may contain detergents, surfactants),
● Some dispersants can be in themselves toxic! – Such as Corexit 9500, which is
banned in the UK,
● Sites are then stimulated through the addition of nutrients, such a carbon,
nitrogen and water,
● In some cases, bioaugmentation is used where known microbial fuel degraders
such as the well-established Proteobacteria Pseudomonas sp is also added
 Advantages (of Bioremediation) Disadvantages

Costs: as low as $75/cubic yard Scale-up from bench/pilot scale to the

compared to conventional methods like field.Site-specific and chemical specific
incineration or secured land filling at limiting factors required
$200-$800/cubic yard

In situ: Minimal site disruption, reducing Failure of regulatory agencies to consider

public concern it an option

Relative simplicity of the technology Development time and costs. Often

compared to traditional approaches slower than methods such as incineration
- particularly when substrate or nutrient
levels diminish. Microbes may switch to
an alternate energy sources

Operational requirements may be lower Liability disputes if the degradation

than say on-site incineration, soil washing process ceases when the contaminant
systems etc levels are still above established goals

Bioremediation in temperate environments

● Deep Water Horizon Oil Spill
- When: April 20, 2010
- Problem: Largest marine oil spill in history of the petroleum industry, killing
eleven people.
- 4.9 million barrels of oil was discharged
- Extensive damage to marine and wildlife reported
● Stimulation and bioaugmentation
- Containment and the dispersant Corexit (7,000 m3) was used combined
with mixing
- This dispersant was potentially toxic (endocrine disrupting chemicals)
- Naturally present microbes (Colwellia sp) consumed part of the oil
- Alcanivorax borkumensis was added to aid the process
- In 2014, BP claims clean-up complete

Bioremediation of cold environments

● Rarity and susceptibility of polar soils
- Less than 0.4% of Antarctica is ice-free
- Wildlife, human activity and contamination concentrated in ice-free areas
- Time required to remediate is much longer than temperate environments
 ● The need to remediate:
- Management of wastes & contaminated sites in Antarctica are subject to
the Protocol on Environmental Protection to the Antarctic Treaty 1991
(Madrid Protocol)
- Stipulates “signatory Treaty Nations assess, minimise, remediate &
monitor possible impacts of their activities on the Antarctic environment”

New approaches being used by the Australian Antarctic Division for soil
bioremediation
● Site 1: SubAntarctic Macquarie Island
- Diesel Fuel Contamination (800-27,000 mg kg-1)
- Limited by low nutrients & temperatures; low water & oxygen availability
Bioremediation via biostimulation:
- Air sparging and nutrient addition started in 2009
- Community recovery associated with bioremediation
- TPH going down
- Nitrification species appearing
- Increase in diversity
But…
- Bioremediation was variable across the site
- Plateaued at 1,000 mg/kg soil – Future: is 1, 000 mg/kg Ok?
- Alternative approaches such as biopiles or composting to be investigated
● Site 2: Casey station, Antarctica
Engineered biopiles & nutrient amendment
- In 1999, a fuel spill released 10,000L of diesel fuel in 1,700 tonnes of soil
and rock
- In 2005, risks to the local environment were too high to continue by natural
attenuation
- In 2011, 600 m3 of contaminated soil was excavated and used to
construct six biopiles
- Biopiles were engineered with a composite barrier system to contain the
excavated fuel-contaminated soil and leachate
- Biostimulation was achieved through annual mixing and the one-time
application of mineral fertilisers.
- Bioremediation successful to 1,000 mg/kg soil
Considerations
- We need Antarctic specific guidelines for remediation target development
- Is 1,000 mg hydrocarbons/kg soil Ok for soil reuse?
- What types of hydrocarbons are in the “unresolved complex mixture” and
are they toxic?
 - If toxic, can we restart the bioremediation process?

Conclusions
● The problem: contamination of the environment by chemicals (Xenobiotics)
● The solution: bioremediation uses living organisms to degrade hazardous
materials into less toxic ones
● Three main strategies exist for the cleanup of contaminated sites
● In both temperate and cold environments bioremediation of oil has been
successfully applied
Lecture 22: Astrobiology
What is Astrobiology?
The study of the origin, evolution, distribution and destiny of life in the universe
● How does life begin and evolve?
● Does life exist elsewhere in the Universe?
● What is the future of life on Earth (and beyond…)?

Astrobiology Goals
To understand:
1. How life arose on Earth (organisation of matter into living systems)
2. How life evolves at the molecular, organism, ecosystem levels
3. How the terrestrial biosphere has co-evolved with Earth
4. The limits for life on Earth
5. How to recognise the signature of life on other worlds
6. If is there is (or once was) life elsewhere in our Solar system

What is Life?
● “The condition that distinguishes animals and plants from inorganic matter,
including the capacity for growth, reproduction, functional activity, and continual
change preceding death.”

Properties of Life
● Order
● Reproduction
● Growth and development
● Energy utilisation
● Environmental responses
● Evolutionary adaptation
● Evolution is the key to ensuring the continuance of life on Earth

Environmental requirements for life

● Source of elements and molecules from which to build living cells – building
blocks
● Source of energy for metabolism
● Liquid medium for transporting molecules of life

Liquid as a medium for life

● Life (as we know it!) comprises of chemical interactions that take place in the
liquid state
● Functions of a liquid in cell survival:
 - organic molecules need to be able to ‘float’ to thus come in contact
- liquid transports chemicals and waste products to/from cells
- ability to dissolve some solutes while others resist - boundaries
- medium that provides both upper/lower temp and pressure limits at which
biochemical reactions occur

Why Water?
● Hydrogen bonding raises freezing and boiling points of H2O
● Water plays a key role in many reactions (e.g, respiration photosynthesis)
● Water acts as a climatic stabiliser
- Frozen water floats
- insulation for life below
- Self-shielding against UV radiation (ozone)
● Water ideal solvent for proteins
- stability/solubility in folded state

Cells
● Basic units of Life
● Matter inside separated from outside by membrane
● Many similarities shared across domains
● Suggests common ancestor

Carbon Based Life

● Life on Earth made from 20 different elements
● However C, O, N, H comprise 96% of living mass
● Life is effectively carbon based
● Why?
- Strength in chemical bonds - 4 atoms at a time
- Most versatile building blocks of molecules
- Variation in carbon skeletons contributes to diversity of organic molecules
and thus diversity of life

Molecular components of cells

● Carbohydrates - provides energy, cell structure
● Lipids - energy stores (fats), membrane components
● Proteins - structural components, enzymes (catalysts)
● Nucleic acids - hereditary material for all life on Earth

Mutations and evolution

● Process of DNA replication both rapid and accurate - however errors do occur
 ● Mutations can:
- have no effect
- be lethal
- improve the functioning of a cell
● Process of mutation leads to variations among species Mutations thus the basis
for evolution - natural selection
● ‘Without it we would still be anaerobic bacteria and there would be no
music’…..Lewis Thomas

Origin and Evolution of Life on Earth

Origin of Life on Earth:

genesis?
Transition from chemistry to biology

Origin of Life on Earth: panspermia?

● conditions need to be right
● potentially many failed attempts
● however….time is on our side :)

Evolution of Life on Earth

Extreme environments
● salinity
● pH
● pressure
● temperature
● radiation
● unexpected substrates
● combination of above ...

Radiation resistance
● Deinococcus radiodurans are able to grow at core of nuclear reactors
● Can survive 1000 times more radiation than humans!
● Capable of complex DNA repair: entire chromosome destroyed by radiation then
reassembled within hours
● Understanding these repair mechanisms could enhance knowledge of DNA
repair in humans

Life at the extreme

● life survives under conditions previously thought impossible
● extreme measures of adaptation
● What constitutes an ‘extreme’ environment?
● extreme for whom….?
● limits of life ‘as we know it’
● concept of boundaries for life altered forever
● life elsewhere may also be found in broad range of environments

‘Extreme’ organisms and life elsewhere

● Expands range of candidates for life elsewhere How life may survive extended
time in space (e.g., radiation)
● Microbes shown to survive dormant for millions of years
 ● Studying ‘extremophile’ growth will provide insight into developing methods to
resuscitate/culture extraterrestrial life IF it is ever found…

Life Elsewhere?
● Look for water!
● Mars???

Is Mars alive now……?

● Suggestion that liquid water has flowed in the last decade
● New deposits observed consistent with liquid water
● Could there be an accessible subsurface Martian biosphere?
● ‘Lab-on-a-chip’
● ‘Holy grail’ - sample return

Life as we do NOT know it…..?

● Source of elements and molecules from which to build living cells
● Source of energy for metabolism
● Liquid medium for transporting molecules of life

Elements: what about silicon based life?

● Silicon can also form four bonds at once, as carbon
● However……
- bonds are significantly weaker
- silicon rarely forms double bonds
- silicon structures cannot exist long in H2O
● Life based on silicon biochemistry would be more plausible in conditions very
different to Earth

Alternatives to H2O as solvents for life

● Ammonia
- Potential life-sustaining solvent
- Metabolism in liquid ammonia with C=N units conceivable
- Liquid ammonia could present opportunity for life in colder bodies
- However, ammonia less viscous, lower surface tension, smaller
temperature range, and cannot co-exist with free oxygen
● Organic solvents (methane/ethane)
- Abundant in universe
- Protectant against UV radiation
- Biochemistry of organisms would have to be much different (silicon?)
Alternative energy sources
● Electromagnetic wavelengths/fields - conversion of radio waves into chemical
energy; extracting free energy from magnetic fields
● Thermal energy gradients - thermosynthesis could allow membranes to convert
heat into electrical energy during temp cycling
● Osmotic gradients - Coupling movement of water molecules through a
membrane to formation of high energy bond (eg, ATP)
- A hypothetical organism could move between layers of different salinity
and harvest energy

Life elsewhere
● Life cannot be excluded from habitats very different from those of Earth, as a
different set of complex chemical interactions requiring different molecular
components, solvent systems, and energy sources become possible at
temperatures, pressures, and chemical compositions very different from those on
Earth (Schulze-Makuch and Irwin, 2006)
● Is natural selection universal?
● Could the universe be filled with failed life experiments?
● Is intelligence inevitable?

Stromatolites
● Present on Earth > 3.5 billion years
● Geobio systems produced by activity of microorganisms - very complex and
dynamic communities
● Linked to oxygenation of early atmosphere
● Fossil stromatolites are our earliest record of life on Earth
● Earth’s oldest known ecosystems
● ‘Living rocks’

Stromatolites and astrobiology

● Study of life on Earth: Study molecular record in living organisms and geological
record in fossils (mats/stromatolites)
● Understand our own origins: Study processes of co-evolution of our own early
microbial biosphere and its environment
● Possibility of life elsewhere: Stromatolites on Earth may be key in our attempts to
identify and recognize life elsewhere in the solar system biomarkers
● Ability to identify life-sustaining environments beyond our own solar system:
Interpret infrared spectra of distant planetary atmospheres for possible
contributions from geological processes and biospheres
Lecture 23: Clinical Genomics
OUR GENOME, OUR HEALTH
● Variation in our DNA underpins most diseases and influences how we respond to
virtually any medical condition or treatment.
● Interpretation of genomic data enables highly precise treatment of symptoms.
● Genomic information has already had a huge impact on clinical care, particularly
in rare disease and cancer.
● Rapidly declining costs of genome sequencing will enable population-wide
access to genomic data in 10-20 years.
● Genomics is set to transform healthcare (and society) as we know it today
GENOMIC MEDICINE: WHAT ARE THE OPPORTUNITIES?
1. Genetic disease. Diagnosis of rare and inherited disorders.
2. Cancer. Molecular stratification to guide treatment (precision oncology).
3. Disease prevention and treatment personalisation. Predisposition testing, carrier
testing, optimisation and avoidance of adverse drug reaction

HEALTHCARE IMPACT: TRANSFORMING DIAGNOSIS OF RARE DISEASE

● ~1-2% of individuals are born with severe genetic disease - majority remain
undiagnosed
● Diagnosis can guide treatment, patient management, reproductive choices,
future certainty
● Genome sequencing achieves diagnostic rates of >50%
● Implementation of the test for severe disease diagnosis can already make vast
savings to the health system by avoiding expensive unnecessary

Rare Conditions
● Although individual conditions are rare, the aggregate of all rare conditions is
significant.
● Conservative estimates are that 6% - 8% (1-2% severe) of Australians have a
rare condition, and 80% of these conditions are genetic in origin.

THE CHALLENGE OF GENOMIC DIAGNOSIS

● 6.4 billion nucleotides, ~21,000 protein coding genes
● In every individual;
- ~4 million variants
- ~100 protein-damaging variants
- Which one caused the patient’s disorder?
- Which ones predispose to future disease risk?
- Which ones could cause adverse drug reactions?
USING GENOMICS TO DIAGNOSE MENDELIAN DISEASE
● Mendelian diseases are caused by defects in single genes and tend to be rare
● Genetic pathologists use a variety of tools to filter between the millions of natural
variations in the genome to identify the one that is pathogenic (disease-causing)

GENETIC PATHOLOGY OF RARE DISEASE IS LIKE SOLVING A MURDER

MYSTERY
● Multiple lines of evidence are gathered and interrogated
● Was the accused capable of committing the crime?
- Is the gene associated with the phenotype? i.e. could damage to this gene
cause the disease?
● Does the injury match the weapon?
- Does the genetic variant damage the gene? i.e. does it create a premature
stop codon or would it damage the active site of the protein to stop it
functioning?
● Has the weapon been used before in a crime?
- Has this genetic variant been associated with this disease previously in
the scientific literature?
● Does the accused have a criminal history?
- Does the inheritance pattern of the disease fit with incidence of the
variant? e.g. if the condition is recessive, are both parents carriers?
● Was the accused picked out in a line up?
- Is the variant rare in the population? Common variants are not normally
disease-causing

THE FUTURE: GENOME-INFORMED HEALTHCARE

● Genomic information will grow in its applicability to inform healthcare across
increasingly diverse health specialities;
- i.e. from paediatrics and rare disease, to cancer and cardiology, to
nephrology and neurology, to ophthalmology and orthodontics, to general
practice and lifestyle.
- In parallel, the capacity for genomics to detect disease early, avoid
adverse reactions or prevent disease will grow.
● The value of genomic information in most disease contexts will only be realised
in the context of other clinical information.
● Genome-Informed Healthcare enables clinical decision-making to occur in the
context of an individual’s genome.
IMPRECISION MEDICINE
● Much of the medical literature and most clinical trials are undertaken in
Caucasian populations
● Genotype-phenotype associations and allele frequency models are largely
inferred from reference databases that primarily comprise Caucasians
● Precision medicine may not be so precise if ethnicity is not considered
● Exclusion of ethnic groups from certain treatments can also lead to increasing
inequality within the population

GATTACA: HOW CLOSE ARE WE?

● Human genome sequencing is rapidly becoming commoditised.
● Limitations remain in some key areas:
- Editing of the genome (small number of changes)
- Variant interpretation with variable expressivity and penetrance
- Poorly understood genetic contributors in complex disease
- Multiple contributors to complex traits such as height
- Confounding factors of environment
● Genetic engineering of human embryos is illegal
● If we alter a gene to achieve a particular purpose, could there be undesired side
effects?
● Societal divisions, health disparity, loss of diversity…
Lecture 24: Bioinformatics and Microbial genomes
Technology assists research in biology but generates large amounts of data
● “Over the past few decades, major advances in the field of molecular biology,
coupled with advances in genomic technologies, have led to an explosive growth
in the biological information generated by the scientific community. This deluge of
genomic information has, in turn, led to an absolute requirement for
computerized databases to store, organize, and index the data and for
specialized tools to view and analyze the data.” – National Center for
Biotechnology Information USA (NCBI)

What is Bioinformatics?
● Bioinformatics is:
- The field of science in which biology, computer science, and information
technology merge to form a single discipline. - – National Center for
Biotechnology Information USA (NCBI)
- A branch of biological science which deals with the study of methods for
storing, retrieving and analyzing biological data, such as nucleic acid
(DNA/RNA) and protein sequence, structure, function, pathways and
genetic interactions. - Wikipedia
- The emerging field of science which grows from the application of
mathematics, statistics and information technology... to the study and
analysis of very large biological, and particularly genetic, data sets. – W.
Ewens & G. Grant 2001
● Activities in bioinformatics often emphasise the engineering but technologies are
based on science

In the case of bioinformatics: What is the science of data analysis?

● Statistics: this field gives us methods for analysing data
● Computers are a technology for implementing methods and models
● Phylogenetic methods make assumptions about how sequences evolve.
● Parametric statistical methods like t-tests assume underlying measurements are
normally distributed

Some conclusions
● Biotechnology is producing a flood of data
● Computers increasingly play a large role in biotechnology
● Science needs to catch up with technology to make sense of these data
● Software packages implement methods
● Methods usually depend on models
● Models are often mathematical and/or statistical in nature, and they depend on
assumptions about data
● Analysing flood of data properly will require a deep understanding of processes
underlying data
Glossary

3 domains of integral membrane proteins - extracellular (hydrophilic), intercellular,

transcellular (proteins that span the membrane but have different domains on each side
of the membrane)

4 main types of macromolecules - proteins, carbohydrates, lipids, nucleic acids

active transport across the membrane - moves molecules against their concentration
gradient

allele - a varied form of a gene

alphabet - must be able to be copied and passed on to progeny and readily accessed
for the information it contains

anabolism - consume energy to build complex molecules from simpler ones, the
synthesis of protein from amino acids

anaphase - spindles pull the chromosome apart into daughter chromosomes

antibiotic targets - bacterial transcription and translation

apoptosis - programmed cell death removes unwanted cells during development and
removes damaged cells throughout life

asexual reproduction - produces offspring that are genetic copies of the parents

ATP - the energy shuttle of the cell

ATP composition - ribose (sugar), adenine (nitrogenous base), and three phosphate
groups

ATP hydrolysis - the bonds between the phosphate groups of the ATP are broken using
water, releases energy

ATP synthase location - at the active sites in ball on stick structures

autotrophs - produce organic molecules from CO2 and other inorganic molecules from
the environment

autotrophs energy source - only CO2 as a carbon source

carbohydrates on membranes - an extracellular side so messages are recognised by

other cells

catabolism - release energy by breaking down complex molecules into smaller

compounds

catabolism energy source - the chemical bonds in the large substrate that the enzyme
breaks down

causes of mutation - errors in the nucleotide interpretation, chemically induced DNA

damage, radiation induced DNA damage, insertion of transposable DNA

cells - smallest unit of life that can perform essential activities

cellular respiration - the breakdown of glucose in the presence of oxygen

central vacuole in plants - acts as ion repository

chemiosmosis - the respiratory chain pumps protons out of the mitochondrial matrix into
the inter membrane space creating a proton gradient

chemotrophs energy source - chemicals

chlorophyl - found in chloroplasts are pigments required to carry out photosynthesis

chloroplast membranes - outer and inner membrane separated by inter membrane

space

chloroplasts inner membrane - more selectively permeable

chloroplasts interior - a dense gel-like substance called the stoma

chloroplasts location - all green parts of the plant

chloroplasts outer membrane - quite pores

cholesterol in the membrane - helps to make it more solid

compartmentalisation - cellular structure to help bring order to metabolic pathways

components of electron transport chain - all proteins, located in the inner mitochondria
membrane

control of respiration - feedback inhibition of allosteric enzyme

cycles of DNA amplification - each cycle doubles the number of DNA molecules
exponentially

cytokinesis - cytoplasm divides for formation of 2 daughter cells

cytoskeleton components - microtubules, microfilaments, and actin filaments

cytoskeleton function - support and motility

dark reaction location - the stoma

denaturation - loss of the active site due to unfolding

diffusion across the membrane - some molecules can cross the lipid bilayer down the
concentration gradient

disaccharides - consist of two monosaccharides: joined by glycosidic linkage or

glycosidic bond

DNA packaging - DNA is complexed with proteins to form chromatin which is folded and
coiled into chromosomes that help regulate activity of genes

DNA structure - double helix structure of repeating sugar - phosphate units containing
nucleic bases: A, C, G, T

duplication - can lead to repeat sequences

endocytosis - the uptake by invagination of the membrane

endosymbiotic theory - some organelles (mitochondria and chloroplasts) originated from
the uptake and symbiosis of single celled microorganisms

energy requirements - in order to survive organisms must obtain energy and a carbon
source from the environment

enzymes - a type of protein that acts as a catalyst to speed up chemical reactions by

lowering activation energy

epistasis - interaction between loci, sometimes the expression at one locus depends on
the genotype at another locus

excited electrons - elevated to a state with more potential energy

facilitated diffusion - channels form holes in the membrane and make a direct
connection between what is outside and what is outside

feedback inhibition - the product of a pathway inhibits an enzyme at the start of the
pathway

fission - break apart

founder effects - if the founders of a location have a certain condition, the results in a
high prevalence of that condition in coming generations

frameshift - gives a completely new amino acid

function polypeptide - consists of one or more polypeptides which are precisely twisted,
folded, and coiled into a unique shape

fusion - two chromosomes join together

generation of ATP - oxidative phosphorylation and substrate level phosphorylation

genetic drift - changes in allele frequencies due to chance events in small populations

genetics - the study of heredity and how biological information os passed from
organisms to their offspring

genome - the complete genetic composition

genotype - the sequence of alleles

glycogen - stored by animals mainly in the liver and muscle cells (hydrolysis of glycogen
releases glucose when the demand for energy increases, cannot sustain energy needs
for a long period of time)

glycolysis - found in all organisms and consists of 10 enzyme - catalysed reactions

glycolysis location in eukaryotes - the cytoplasm

glycolysis pathways - energy investment and energy payoff

golgi apparatus contents - consists of cistern: flattened membranous sac with

directionality

golgi apparatus function - proteins are modified, stored and transported onwards from
here, new vesicles form and leave to new sites

heterotrophs - obtain organic molecules through other organisms e.g. humans

heterotrophs energy source - one or more organic carbon sources (e.g. glucose)

high energy phosphate bonds - link the ATP phosphate groups, are relatively unstable
and very high in energy which can be hydrolysed and released to perform cellular work

human fuel metabolism - fats and amino acids can only be broken down by catabolic
pathways that involve respiration and conversion ultimately to CO2 and water

incomplete dominance - two alleles contribute to the phenotype

initial membrane structure - made inside the endoplasmic reticulum

insertion or deletion - one or more nucleotides into or out of the sequence

inside the stoma - membranous sacs called thylakoids

intermediate filaments - fibrous proteins coiled into cables that assist with maintenance
of cell shape, anchorage, and formation of nuclear lamina
interphase - growth and replication of cellular components, duplicated chromatin is
tangles in the nucleus

invagination - the massive reorganisation of the embryo from a simple ball into a
multilayered organism

inversion - part of the chromosome breaks off and flips back to front

ion trasport and nervous stimulation - the cells become excited causing sodium
channels to open making the inside of the cell more positive and increasing membrane
potential

leading strand synthesis - started to be synthesised by DNA polymerase after the RNA
primer is made

light reaction location - the thylakoids

light reaction process - water is split and light is absorbed by chlorophyll pigments
powering the transfer of electrons hydrogen and NADP+

lipid rafts - mechanisms that can be used for movement across the membrane

lipids - hydrophobic: insoluble in water due to non-polar covalent bonds of carbon and
hydrogen

lipids function - perform energy transport and storage, major structural component of
cell membranes (phospholipids)

locus - a place on the chromosome where a gene is located

lysosome function - hydrolyse macromolecules, breakdown lipids, carbohydrates, and

proteins into small molecules for use in the cell, and remove junk and clutter in the cell

lysosomes - enzymes in an acidic environment segregated by the membrane

macromolecule definiton - a large organic molecule formed by the joining of smaller

molecules, usually by a dehydration reaction

membrane permeability - barrier to most molecules, important for maintenance of cell

integrity
membrane potential - voltage difference across a membrane created by the difference
in the distribution of positive and negative ions

mendel's first law - diploid individuals have one copy from mum, one from dad

mendel's second law - for two genes, the pairs of alleles assort independently into
gametes

metabolism definition - the totality of an organism's chemical reactions

metabolism pathways - catabolic and anabolic pathways manage the material and
energy resources of the cell

metaphase - chromosomes line up the the middle of the cell and spindles attach

microfilaments - two intertwined strands of actin that assist in maintenance of cell shape
and muscle contractions

microtubules - hollow tubes that maintain cell shape, cell motility, chromosome
movements in cell division and organelle movement

missense mutation - results in a change in amino acid identity

Mitochondria function - carry out respiration and reduce oxygen to water, found in
muscles and used for aerobic activity

mitochondria: inner membrane - semi-permeable with folded structure for maximum

surface area

mitochondria: inter membrane space - has mitochondrial DNA and ribosomes

mitochondria: outer membrane - moves things across quite easily

mitotic phase - nucleus divides and chromosomes are distributed to daughter cells

molecules of life - CHNOPS: carbon, hydrogen, nitrogen, oxygen, phosphorus and

sulfur
monosaccharides - simplest form of carbohydrates (e.g. glucose): can be used for fuel
and be combined into polymers

mutagens - agents that cause mutations

mutation definition - change in the nucleotide sequence of an organisms DNA creating

genetic diversity

no effect mutation - results in a different codon that prescribe the same amino acid

non-disjunction - homologous chromosomes don't separate during meiosis

nonsense mutation - results in a new stop signal

nucleic acids - polymers of nucleotides (DNA and RNA): DNA directs protein synthesis
via RNA

oxidation - loss of electrons

oxidation - reduction relationship - if one molecule is oxidised, another one is

simultaneously reduced

oxidative phosphorylation - inorganic phosphate is added to ADP to produce ATP,

process powered by the redox reactions of the electron transport chain

PCR has a negative charge - the smaller shorter DNA molecules can be pulled through
electrophoresis towards the positive charge faster

peptide bonds - backbone between amino acids

peptides - polymers of amino acid monomers that fold into a specific 3D structure to
make proteins

peroxisomes - specialised metabolic compartments that carry out reactions that produce
hydrogen peroxide

phagocytosis - cellular eating: cell engulfs the particle by wrapping pseudopodia around
it and packaging it into a large vesicle or vacuole

phenotype - the physical trait or gene being expressed

photoautotrophs - organisms that synthesise organic molecules from mostly CO2 and
H20

photorespiration - ATP is used and no sugar is generated in this process

photosystem I - the transfer of electrons is not coupled with the transfer of protons
across the thylakoid membrane

phototrophs energy source - light from the sun

pigments - substances that absorb visible light

pinocytosis - cellular drinking: proteins can diffuse across the membrane into the
vesicles

pleiotropy - one gene can affect multiple traits

polygenic traits - traits influenced by many genes

polymers - formed by covalent linkages of smaller units called monomers

polyribosomes - mRNA can be used to synthesise many proteins simultaneously

polysaccharides - composed of many monosaccharides: act as energy storage and

transport molecules

prophase - chromosomes are formed and early mitotic spindle forms

protein shape - 3D shape crucial to function of the enzyme and determined by the
sequence of amino acids

proteins function - related to shape and chemical properties of their monomers,

polymers of amino acids with diverse structures and function

proteins primary structure - the sequence of amino acids

proteins quaternary structure - overall protein structure from two or more peptide
subunits (only if the protein is composed of more than one polypeptide)
proteins secondary structure - folding or coiling the peptide into a repeated configuration

proteins tertiary structure - the overall 3D shape of the polypeptide

proton motive force - the imbalance of protons represents a source of free energy

pyrimidine dimer formation - repairs DNA damage

pyruvate in fermentation - converted into waste products

pyruvate in respiration - converted to acetyl-CoA in the mitochondrion

receptor mediator endocytosis - cells have special proteins at the surface that bind to
specific molecules

redox reactions - oxygen is loss, reduction is gain (OILRIG)

reduction - gain of electrons

replication first step - separation into two parent strands that serve as templates for the
order of nucleotides along the new complementary strand

requirements of DNA polymerase - must have a 3' OH group to add on to, will only
elongate DNA in the 5' to 3' direction, and can't initiate DNA synthesis unless there is a
primer that contains a 3' OH group

result of sexual reproduction - produces offspring that are a genetic combination of both
parents

ribosome - the protein synthesis factory

ribosomes function - make proteins that can be used in the cytoplasm

role of the mitochondrial membrane in ATP synthesis - the transport of H atoms along
the respiratory chain depends on the fluidity of the membrane to allow different protein
components to move and interact

rough endoplasmic reticulum (ER) - surface associated with ribosomes

saturated fatty acids - contain single carbon-to-carbon bonds

semi-conservative - combines the old and the new

sidedness - asymmetrical distribution of proteins, lipids, and carbohydrates between the

two sides of the membrane

simple sequence repeats - serve no function but are simply present in the DNA,
repetitions of the same amino acid

single-base changes - one base in the genetic code is changed

small population effects - the smaller the population size, the bigger the effect of
mutation

smooth endoplasmic reticulum (ER) outer surface - lacks ribosome and plays a diverse
role in metabolic processes e.g. drug detoxification and lipid storage

sodium-potassium pump - electronic pump generates concentration gradient of sodium

and potassium and contributes to membrane potential in all animal cells

sources of genetic variation - mutation, sexual reproduction, recombination

stages of mitotic cell division in animals - interphase, prophase, metaphase, anaphase,

telophase and cytokinesis

stages of transcription - initiation, elongation, termination

starch - stored by plants as granules with various cellular structures (stored energy can
be later accessed by hydrolysis)

steps of the dark reaction - carbon fixation, reduction, regeneration

stomata - microscopic pores through which CO2 enters and oxygen exits

stomata activity in heat - close to prevent water loss, limiting access to CO2

storage polysaccharides - starch and glycogen

substrate level phosphorylation - an enzyme transfers a phosphate group from a

substrate molecule to ADP
telophase - new nuclear envelope forms

the dark reaction - CO2 incorporated into organic molecules in carbon fixation and
electrons from NADPH are used to reduce this along with energy in the form of ATP

the endomembrane system function - regulates protein traffic and performs metabolic
functions

the genetic code (triplet code) - triplet code is redundant as it codes for 20 amino acids

the origin - the point at which DNA replication begins

the respiratory chain - reduced cofactors transfer their reducing power to oxygen
through a series of redox reactions

the start codon - AUG always starts the amino acid chain

the TCA cycle - is acetyl-group of acetyl-CoA is broken down inside the mitochondria

thylakoid membrane systems I and II - linear electron flow drives ATP synthesis and
NADPH formation

transcription elongation - the polymerase moves downstream unwinding the DNA and
elongating the RNA transcript and then DNA reforms the double helix

transcription initiation - RNA polymerase binds to the promoter region upstream of the
gene, DNA strands unwind, RNA synthesis is initiated by the RNA polymerase, RNA
polymerase makes a complementary RNA copy of the DNA sequence

transcription termination - RNA transcript is released, the polymerase transcribes a

terminator sequence to signal the end of a gene and the polymerase detaches from the
DNA

translation - reads the tRNA and pairs it with amino acids

translation elongation - codon recognition, peptide bond formation transfers the growing
chain from the A site onto the amino acid at the P site
translation initiation - small subunit binds the mRNA and initiator tRNA, large subunit
binds with the initiator tRNA in the P site, energy goes from molecule ATP to molecule
GTP

translation termination - when a stop codon occurs in the mRNA it is recognised by a

release factor causing the last tRNA to leave the ribosome and the newly synthesised
protein is released

translocation - part of the chromosome breaks off and relocates

tRNA - one end has an amino acid and the other end has a triplet code

two processes of photosynthesis - the light reaction and the dark reaction

types of carbohydrates - monosaccharides, disaccharides, and polysaccharides

types of mutation changes - no effect, missense, nonsense, frameshift

types of mutations - single-base changes, insertion or deletion, duplication, fusion,

fission, inversion, translocation

unsaturated fatty acids - contain one or more double bonds (makes it more fluid)

vacuoles - vesicle that contains an environment different to the cytosol that differs
depending on their role

vacuoles in unicellular eukaryotes - remove water to maintain ion concentration

visible light spectrum wavelengths - 380-750 nm

wavelength and photon relationship - the shorter the wavelength, the greater the energy
of each photon