MICROBIOLOGY

EUKARYOTES vs PROKARYOTES:

• EUKARYOTES (animals, plants, protists, fungi)

o Nucleus (DNA+histones)
o linear chromosomes
o cytoplasmatic organelles
o 10 – 100 µm = larger
o Mitosis

• PROKARYOTES (bacteria, archaea)

o no nucleus
o one nucleoid containing circular DNA
(chromosome) + extrachromosomal DNA
(plasmids)
o no cytoplasmatic organelles
o 1 – 10 µm = smaller
o Higher reproduction rate, cell division is simpler and high mutability

Both have cell mb, DNA as a carrier for genetic information, ribosomes, and basic metabolic pathways

MICROORGANISMS

• bacteria (1 - 10 µm)
• archaea (1 - 10 µm)
• fungi (10 - 100 µm) = eukaryotes, cell wall from chitin, grow in hyphae or as single cells (yeast),
reproduction via spores, usually harmless, mushrooms (fruiting bodies of higher fungi), moulds
(toxic), Saccharomyces cerevisiae (baker’s yeast), Pichia pastoris, Penicillum camemberti (cheese)
• viruses (1 nm - 1 µm) = genetic material protected by a protein cover, no metabolism = DNA/RNA
enclosed in a protein coat (no cellular structure & no metabolism). They rely on host cells to
replicate
• prions (1 nm) = not living, proteins causing disease, transmittable, BSE, CJD, scrapie
• helminths (worms) - (10 - 100 µm) = eukaryotes (animals), as infective stages (eggs) are
considered MOs = live as endoparasites (within a host), flatworms (tapeworms), roundworms
(hookworms)
• protists - (10 - 100 µm) = unicellular eukaryotes, usually harmless

STRUCTURE OF A BACTERIAL CELL

Bacterial shapes:

• coccus (streptococci, staphylococci, diplococci)

• rod (bacillus, streptobacilli, diplobacilli)
• spirillum, spirochete
• hypha
• filamentous
• vibrio

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Bacterial cell components:

• cell membrane
• cell wall
• nucleoid
• 70S ribosomes
• pili
• flagella
• capsule
• endospores
• storage granules

Cell membrane:

• surrounds the cell material

• selective barrier (only certain molecules can pass like small,
hydrophobic, e.g., O2/CO2 and not large or charged molecules
= they require assistance of transport proteins)
• contains phospholipid bilayer with embedded proteins = like
eukaryotic membrane but bacterial one does not contain
cholesterol
o Phospholipids have hydrophobic (water-fearing) tails and hydrophilic (water-loving)
heads, which arrange themselves in two layers.

DNA (nucleoid) = genetic material:

• 1 circular chromosome that aggregates to form the nucleoid

• most bacteria contain also smaller circular DNA molecules –
plasmids – serve as tools in molecular biology as they are easily
manipulated and introduced into bacteria. Plasmids can confer an
advantage to bacteria (e.g., for AB = transfer of resistance genes
between bacteria)

Ribosomes: (smaller than eukaryotic, 70s)

• protein synthesis machines (translation)

• 5 000 – 50 000 ribosomes per cell
• 70S: large 50S subunit, small 30S subunit
• some antibiotics block bacterial ribosomes but not eukaryotic ones
(aminoglycosides, tetracyclines, macrolides)

Cell wall:

• strong, rigid, pressure-resistant net around the cell membrane

• ➜ structural support and protection
• consists of a peptidoglycan (murein):
o peptide: amino acids (connect glycan chains)
o glycan (sugar): N-acetylglucosamine (NAG), N-acetylmuramic acid (NAM)(form backbone)
crosslinked by short peptide chains.
• lysozyme: enzyme that is found in egg whites, tears, saliva = can digest cell walls; protection
against bacterial infections

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• some antibiotics block formation of bacterial cell wall such as such as B-lactams (penicillin) and
vancomycin (Mycoplasma lack bacterial cell wall and thus have a resistance against AB).
• according to structure of a cell wall, bacteria can be divided into: Gram positive or Gram negative
• under microscope, both look the same (Mycoplasma and
related bacteria do not belong here)
• GRAM STAINING: we can stain these bacteria to see in which
group they belong via staining solutions such as crystal violet,
iodine treatment, ethanol (decolorization) and safranin

a) Gram positive (VIOLET):

• thick layer of peptidoglycan (40 layers)
• teichoic and lipoteichoic acids connect the cell wall with cell membrane
• cannot wash out the color because the layer is thick
• Bacillus (rod-shaped, endospores, facultative aerobic)

b) Gram negative (PINK):

• thin layer of peptidoglycan within the periplasmic space
• surrounded by outer membrane contains porins and LPS
(lipopolysaccharides)
• we can wash out the color because the peptidoglycan is
not that thick
• More resistant to AB. Periplasmic space between cell mb & outer mb is filled with
proteins/enzymes

LPS (lipopolysaccharide) = endotoxin:

• hydrophilic part = sugars (outer part recognized by antibodies)

• hydrophobic part = lipids (activates innate immunity)
• LPS is called endotoxin (role in virulence)
• vaccines are without LPS

Enterobacteriaceae:

• Escherichia coli: Gram negative, rod shaped, motile, facultative aerobe, found in intestines, fecal
indicator (many diseases are transmitted via fecally contaminated water, E. coli is not itself a
pathogen because it cannot survive outside the body for a long time)
• Salmonella and Shigella species: pathogenic (gastroenteritis), transmission via food and water
• Klebsiella, Enterobacter and Proteus species: found in water, soil and intestinal tract

Bacillus:

• Bacillus subtilis, Bacillus cereus, Bacillus anthracis: Gram positive, rod shaped, obligate or
facultative aerobes, form endospores

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Pilli/fimbriae:

• outer extensions, contact with surface and other bacteria

• sex pilli: transfer of DNA

Flagella:

• movement, rotation, gliding

• consists of protein = flagellin (helical structure)
anchored via other proteins
• basal body and a hook in cell membrane (can be stained to distinguish bacteria)

EPS (extracellular polymer substances) = sugar molecules secreted by bacteria enabling

them to bind to surfaces, aggregate into biofilms, protection.

Capsules and slime layers:

• protection, biofilm formation, binding

• consist of sugar polymers:
o homopolysaccharides = DEXTRAN
o heteropolysaccharides = XANTHAN
• SLIME (lots of water)
• CAPSULE (little water)

Biofilms:

• thick layer of bacteria

• tooth plaque, corrosion, catheter, washing machine plaque

Storage granules:

• storing of energy within glycogen, gas vesicles, magnetosomes

Endospores:

• when some bacteria cannot grow or divide anymore, endospores are formed within several hours
(help bacteria to survive in tough conditions)
• endospores have no metabolism, contain high concentrations of calcium and consist of less than
15% of water, contain DNA (High [calcium, dipioconic acid) = protect DNA)
• structures that are resistant to high temperatures (120°C), harsh chemicals, radiation, dry
conditions (unfavorable conditions)
• They are a problem for Sterilization. When conditions are favorable again, the endospore com
germinate & grow into new bacterial cell
• Endospore formation starts with environmental stress (nutrient depletion / exposure to toxins) ➜
thick protective coat around bacterial chromosome ➜ metabolically inactive and can withstand
extreme conditions.
1. vegetative cell asymmetric cell division
2. pre-spore engulfment
3. spore cortex and coat synthesis
4. spore maturation and release

Bacillus and Clostridium (E. coli does not have them)

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 psychrophilic: <20°C
BACTERIAL GROWTH CONDITIONS mesophilic: 30 - 40°C
thermophilic: >45°C
hyperthermophilic: >75°C
• temperature, pH, oxygen, osmotic pressure, light, nutrients…

Temperature:

• every organism has its own optimal growth temperature

o minimum (cannot grow – death)
o optimum (highest, fastest growth)
o maximum (protein denaturation – death)
• many MOs are mesophilic (30°C - 40°C) – optimal growth

pH:

• every organism has its own optimal pH

o acidophiles (pH < 6)
o neutrophiles (pH = 6-8)
o alkaliphiles (pH > 9)
• to adjust the pH of the medium, we use buffers (keep the
pH stable)
• acids denature proteins

Oxygen:

• obligate aerobes (need oxygen to live)

• obligate anaerobes (do not need oxygen to live)
• facultative anaerobes (both, grow faster with oxygen – E. coli)
• microaerophiles (lower concentration of oxygen)
• aerotolerant anaerobe (with or without oxygen)

O2 can be toxic because it can generate reactive oxygen species (ROS) in cells, such as superoxide
anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH) are chemically reactive molecules
containing oxygen that can cause damage to cellular components such as proteins, lipids, and DNA by
oxidizing them.

Cells have developed various mechanisms to prevent or repair oxidative damage caused by ROS, such
as antioxidant enzymes and non-enzymatic antioxidants ➜ e.g. catalase convert these reactive oxygen
species into H2O and O2.

• during metabolic processes, toxic oxygen species can be produced:

o superoxide anion (O2-)
o hydrogen peroxide (H2O2)
o hydroxyl radical (OH.)
• they are all very reactive (react with all kinds of molecules, oxidize and destroy them)
• aerobes are protected against the toxic oxygen via a special enzyme catalase, that converts these
reactive oxygen species into H2O and O2
• H2O2 + H2O2 ↔2 H2O + O2
• anaerobes grow in CO2 incubators or in anaerobic culture jars

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Osmotic pressure / water activity:

It is the pressure that develops in a solution due to the difference in solute concentration between
two solutions separated by a semipermeable membrane. When a microorganism is placed in a
solution with a higher solute concentration (hypertonic solution), water will tend to move out of the
cell, causing the cell to shrink and become dehydrated. On the other hand, when a microorganism is
placed in a solution with a lower solute concentration (hypotonic solution), water will tend to move
into the cell, causing the cell to swell and potentially burst.

• high osmotic pressure (hypertonic environment) removes water and decreases growth
• low osmotic pressure (hypotonic environment) causes water to enter the cell and causes lysis

Nutrients:

• energy source in the form of macromolecules - proteins, lipids, carbohydrates, DNA, RNA
• provided via the growth media
• elements in a bacterial cell: carbon, oxygen, nitrogen, hydrogen and minerals

Hydrostatic pressure:

Particularly those that live in environments such as the deep ocean where the pressure can reach very
high levels. It can affect their growth and physiology in various ways, such as by affecting the fluidity of
the cell membrane, the stability of proteins and enzymes, and the transport of nutrients and waste
products across the cell membrane.

Light

Growth factors: vitamins, aminoacids, purines and pyrimidines and their precursors

GROWTH MEDIA

• solid or liquid preparation for bacterial growth

• liquid (broth)
• solid (plates, tubes, slants) ➜ e.g. Agar plates: polygalactoside
from marine red algae, not degradable by most bacteria, melts
at 100°C, solidifies at ~40°C / Gelatine: proteins from animals,
degradable by most bacteria, melts at 28°C, heat sensitive

• On plates, bacteria form colonies = millions of bacteria formed

from 1 bacterium (all are identical)
• color and shape of colony can give hint, which bacterium it
might be

Two main types of media:

1. DEFINED: exact chemical composition is known, contains salts and purified chemical compounds,
usually colorless
2. COMPLEX: material of biological origin (yeast extract, casein, beef extract, soybean extract,
peptones), usually yellow, bacteria grow faster and better, highly nutritious, differences from
batch to batch (cows from farms or meadows)

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Types of solid media:

1. AGAR: vegan, sugar from marine red algae (powder), not degradable by most bacteria, this
powder is mixed with broth, soft (4 g/l), solid (10-15 g/l)
2. GELATINE: protein from animals, degradable by most bacteria, heat sensitive

Special growth media:

1. SELECTIVE: substance that does not allow unwanted bacteria to grow = growth of desired
microbes
2. ENRICHMENT: increases the growth of desired bacteria, bacteria grow better (similar to selective)
3. DIFFERENTIAL: allows to see the differentiation of desired microbes from others (color change)

BACTERIAL DIVISION AND ITS PHASES

• bacteria divide by BINARY FISSION

• bacteria grow exponentially (semi-
logarithmic scale)

1. DNA replication
2. cell elongation
3. septum formation
4. formation of distinct walls
5. cell separation
6. 2 identical bacteria

= ONE GENERATION

Growth phases of a bacth culture:

• lag-phase – little or no cell division, cells are adapting to

the new environment
• log-phase – exponential growth
• stationary phase – nutrients run out or waste products
aggregate
• death phase – exponential death phase

N0 = number of bacteria at t0

N = number of bacteria at t1

n = number of generations

g = generation time

v = division rate

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Exponential growth?

N = 2n

How many cells after certain number of generations?

N = 2n × N0

How many generations?

𝑵
n = 3,3 × log ( )
𝑵𝟎

How long does it take cells to double?

• GENERATION TIME (g) = duplication time

𝒕 𝒕
g= g= 𝐍
𝒏 𝟑,𝟑 × 𝐥𝐨𝐠 ( )
𝑵𝟎

• high generation time = SLOW

• low generation time = FAST

How fast does my culture grow?

• generations per hour (v)

𝟏 𝒏
v= =
𝒈 𝒕
• high division rate = culture grows fast
• low division rate = culture grows slow

TYPES OF CULTURES

1. batch culture: type of microbial culture that involves the cultivation of microorganisms in a closed
system with a fixed volume of growth medium = raw material and microorganisms are put in the
fermenter/Erlenmeyer flask and system is incubated for a certain time, all the raw material is used
up and there is loads of the product, fermenter is emptied and product purified (ONE BATCH),
fermenter is cleaned out and a new batch can be started
2. continuous culture: continuous harvest of the product and addition of new medium, bacteria are
kept at a certain density and in certain growth phase. = MOs continuously provided with nutrients
and culture can be maintained over extended period.
3. fed-batch culture: after a batch phase, nutrients are added = feed phase → modified form of
batch culture in which additional nutrients or substrates are periodically added during the
cultivation process. Allows for controlled nutrient feeding to enhance growth or product yield
without maintaining a constant culture volume.

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MEASUREMENT OF GROWTH

= how many bacteria/ml are in a culture

A. Direct measurement:

1. counting chamber/direct microscopic count:

• slide with a microscopic grid, defined distance from slide to cover slip,
cells in a defined volume are counted
• advantages: counting high concentrations (107 cells/ml), result without
incubation
• disadvantages: dead cells cannot be distinguished from living cells, time-consuming, low
concentrations cannot be counted, phase contrast microscope, debris, dust, dirt

2. plate count:
• serial dilution, plating (spread plate or pour plate), incubating
& counting colonies, calculate CFU/ml
• advantages: number of CFU (viable Mos), concentration down
to 30 CFU in 100 microliters
• disadvantages: cells grow in chains, clumps or build one
colony, result only after incubation, dilution errors
a) spread plate: sample is pipetted onto agar plate, sample is
spread evenly over the surface of agar by using sterile
spreader, sample is incubated, surface colonies are counted
b) pour plate: sample is pipetted into sterile plate, sterile
medium is added and mixed with inoculum, sample is
incubated, subsurface and surface colonies are counted

𝒄𝒐𝒍𝒐𝒏𝒊𝒆𝒔 × 𝟏𝟎𝟎𝟎6𝒍
CFU/ml =
𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 × 𝒑𝒍𝒂𝒕𝒆𝒅 𝒗𝒐𝒍𝒖𝒎𝒆 (6𝒍)

3. filtration:
• used for CFU of drinking water
• bacteria are larger than pores of filter so we can count CFU
• advantages: number of CFU (viable Mos), very low concentration
• disadvantages: cells grow in chains, clumps or build one colony, result only after incubation

4. most probable number method:

• used for bacteria that do not grow on solid media or with
liquid differential medium

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B. Indirect measurement: turbidity, metabolic activity, weight/cell volume

1. turbidity:
• in UV spectrophotometer usually at 600 nm (OD600)
• the more the MOs, the less the light
• advantages: fast, reliable, calibration possible (how much light is lost)
• disadvantages: only used in certain concentration range (0.1-0.6), dust particles

GROWTH CONTROL

• sterilization: killing, removal of all viable MOs and viruses (sterile)

• decontamination: makes an object safe to handle
• disinfection: elimination of pathogens

Temperature:

1. heat:

= bactericidal (kills bacteria by denaturating proteins)

• moist (boiling, steam – autoclave)

• dry (hot air oven, flaming, incineration)
• pasteurization (mild heat to reduce number of bacteria (milk, juice))

= problem: ENDOSPORES!

• autoclave: high temperature and high pressure, water is heated up over 100°C, high temperature
kills even endospores
• hot air oven
• D-value: decimal reduction time, time in which 90% of original population is killed, 104 CFU … 90%
killed, 10% survived … 103 CFU, different for every treatment and organism

2. cold:

= bacteriostatic (prevents bacteria from further growing)

• +4°C = refrigerator (bacteria that can grow in cold)

• -20°C - -80°C = deep-freezing (used to store bacterial cultures)
• -196°C = liquid nitrogen (used to store bacterial cultures)

Radiation:

• non-ionizing (UV-light): cannot penetrate surfaces (dangerous to eyes and skin)

• ionizing (X rays, gamma rays): used to sterilize plastic ware

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Filtration:

• membrane filters: used to sterilize heat sensitive liquids, filtering bacteria

• depth filters: filtration of air, HEPA filters in cell culture hoods, clean rooms

Chemical methods:

• antiseptics: microbicidal agents mild enough to be applied on skin

• disinfectants: agents that kill microorganisms, not safe to use on living tissues
• sterilants: destroy all forms of microbial life (including endospores)
• alcohols: 50% - 70% ethanol or isopropanol, kills by disrupting the lipids of the cell membrane and
denaturing proteins, concentrated alcohol cannot enter the cell
• halogens: chlorine, iodine oxidizing agents, denature proteins
• surface-active agents: decrease surface tension, soap
• heavy metals
• ozone
• hydrogen peroxide
• formaldehyde

Problems with killing MOs:

= organic material, biofilms, resistant bacteria, pH, temperature, toxicity

BACTERIAL METABOLISM

• oxygenic (aerobic) respiration

• anoxygenic (anaerobic) respiration
• fermentation
• chemolithoautotrophic growth
• photosynthetic bacteria

Metabolism:

• to maintain status quo (cells get old and have to be replaced)

• to grow (cells have to be produced)
• catabolic reactions: break down larger molecules to get energy
• anabolic reactions: new molecules are synthetized to consume energy

ATP:

• adenosine triphosphate
• storage of chemical energy in the cell
• by splitting of the 3rd phosphate, energy is set free and can be used for other reactions
• ATP = ADP + energy
• energy source – chemicals, light
• carbon source – food, light

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1. chemoorganotrophs:
• organic chemicals (glucose, acetate)
• animals, fungi, bacteria
o oxygenic respiration
o anoxygenic respiration
o fermentation

A. OXYGENIC RESPIRATION:
• C6H12O6 + 6 O2 --> 6 CO2 + 6 H2O
• 36 ATPs produced

• redox reactions:
o oxidation: loss of electrons
o reduction: gain of electrons

• electron carriers:
• substances that transfer electrons in reactions from one
molecule to another one
• NAD/NADP/FAD
• NAD transfers 2 electrons in many biochemical reactions
• NADH --> NAD+ (oxidation)
• NAD+ --> NADH2 (reduction)

• respiration:
• glycolysis
• pyruvic acid decarboxylation
• citric acid cycle
• electron transport chain

1. Glycolysis:
• FBP (fructosediphosphate-pathway)
• EMP (Emden-Meyerhof-Parnas-pathway)
• process when glucose is broken down from C6 to 2 C3 atoms (pyruvic acid)
• 4 ATPs are produced and 2 are consumed = we gain 2 ATP FROM GLYCOLYSIS via substrate-level
phosphorylation (energy released during the conversion of organic molecule from 1 form to
another)

• FBP preparatory stage:

• 2 ATP are invested to create FBP, that is later split into 2 C3 molecules
• FBP energy conserving stage:
• each C3 molecule is converted to pyruvic acid
• therefore creates 4 ATP and 2 NADH per glucose (transfer of 4 electrons)

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2. Pyruvic acid decarboxylation:
• from 2 pyruvic acids (C3), 2 CO2 are split off
• remaining C2 (acetic acid residue) is bound to coenzyme giving 2 coenzyme A
• transfer of 4 electrons to 2 NADH2

3. Citric acid cycle:

• TCA cycle (tricarboxylic acid cycle)
• Krebs cycle
• coenzyme A is bound to oxalacetic acid (C4)
• electrons are transferred to coenzymes (NAD, FAD)
• C atoms are oxidized = CO2

= glycolysis and the citric acid cycle not only provide ATP and
reduction equivalents (NADH, FADH) but also precursor metabolites
for building blocks of cell material

• 4 CO2
• transfer of 14 electrons to 6 NADH2 and 2 FADH2
• 2 GTP to produce 2 ATP

SUMMARY:

• glycolysis
• pyruvic acid decarboxylation
• citric acid cycle
• electron transport chain

• in steps 1 to 3, we did not use O2

• respiration produces 36 ATPs
• we have produced only 4 of them so far
• 32 ATPs are missing

4. Electron transport chain:

• electrons of NADH2 and FADH are transferred to the electron
transport chain (membrane proteins with cofactors that can
carry electrons or electrons and protons)
• reduction potential of the electron carriers is increasing
• electrons flow without energy input from carrier to carrier
• terminal electron acceptor is OXYGEN
• electron carriers are arranged in a way that protons are
transported out of the bacterial cell/mitochondrion
• proton gradient (motive force to move flagella – influx of
protons or to make ATP in electron transport phosphorylation)

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• proton gradient: used by ATP synthase to catalyse reaction (ADP to ATP)
• electron transport phosphorylation: oxidative phosphorylation
• ATP synthase: found in bacteria, mitochondria, chloroplasts

result:

= 30 – 32 ATP by electron transport phosphorylation

= regeneration of all NAD and FAD

= O2 takes up electrons and protons and is converted to H2O

SUMMARY:

CARBON ELECTRONS ATP

GLYCOLYSIS Glucose = 2 pyruvic acids 2 NADH 2 ATP
2 pyruvic acids =
PYRUVIC ACID 2 NADH -
2 CO2 + 2 coenzyme A
6 NADH
CITRIC ACID CYCLE 2 coenzymes A = 4 CO2 2 GTP
2 FADH
ELECTRON TRANSPORT
- O2 = H2O 30 – 32 ATP
CHAIN

B. ANOXYGENIC RESPIRATION:
• the same process, but oxygen is replaced by other molecules
• final electron acceptor is not O2 but N2, N2O, H2S, CH4

COMPARISON:

• oxygenic respiration = final electron acceptor in aerobic respiration = more ATP

• nitrate respiration = final electron acceptor in anaerobic respiration = less ATP

SUMMARY RESPIRATION:

= oxygenic respiration requires oxygen

= glycolysis, pyruvic acid decarboxylation, citric acid cycle, electron transport chain

= substrate level phosphorylation and electron transport phosphorylation

= final electron acceptor is inorganic molecule (O2, NO3-)

= yield of ATP/glucose is higher than in fermentation

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C. FERMENTATION:
• only glycolysis (substrate level phosphorylation)
• less electrons = ATP game is much lower
• does not require oxygen
• final electron acceptor is an organic molecule
• electrons, that are transferred to NAD in the first steps of glycolysis, have to be transferred to the
rest of the CARBON BACKBONE OF GLUCOSE:
o to pyruvic acid -> lactic acid
o to some product of pyruvic acid -> other fermentation

= 2 steps:

• glycolysis
• regeneration of NAD

= examples of products: lactic acid, ethanol, CO2, acetic acid, acetone, butyric acid…

2. chemolithotrophs:
• inorganic chemicals (H2, H2S, NH4+)
• bacteria

3. phototrophs:
• photosynthetic bacteria

PHOTOSYNTHESIS:

• harvest of energy from light

• requires light absorbing molecules/pigments (chlorophylls, carotenoids…)
• with light energy, electron in chlorophyll gets excited and is transported in electron transport
chain to NADP
• in this electron transport chain, protons are transferred to the other side of the membrane,
creating a proton gradient
• ATP synthase converts the proton gradient to ATP
• cyanobacteria (Spirulina): obligate photoautotrophs, rely on photosynthesis, gram negative, fix
nitrogen, chlorophyll a and phycobiliproteins, gliding, grow as single cells, colonies or filaments

• oxygenic photosynthesis
• plants, algae, cyanobacteria
• 6 CO2 + 6 H2O ↔ C6H12O6 + 6 O2

• anoxygenic photosynthesis
• green sulfur and purple sulfur bacteria
• 6 CO2 + 12 H2S ↔ C6H12O6 + 6 H2O + 6 S2

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MICROBIAL PRODUCTS

• food industry (cheese, yoghurt, pickles)

• enzymes (amylase, protease)
• polysaccharides (xanthan, dextran)
• nutrients (amino acids, nucleotides, vitamins)
• chemotherapeutic agents (antibiotics)

A. FERMENTATION:
• strict sense: a way to generate energy in which organic compounds serve as electron donor and
electron acceptor and ATP is produced by substrate level phosphorylation
• industrial context: microbial processes carried out aerobically or anaerobically
• cells, cell extracts (yeast, spirulina)
• microbes, enzymes convert substance to desirable product (wine)
• antibiotics
• food additives (amino acids, vitamins)
• chemicals (ethanol, citric acid, acetic acid)

Primary/secondary metabolites:

• lactic acid fermentation

• alcoholic fermentation
• antibiotics

= primary metabolites:

• formed during growth phase = LOG PHASE

• amino acids, nucleotides, fermentation products (EtOH)
• cells consume large amount of sugar in order to produce alcohol

= secondary metabolites:

• formed near the end of growth phase or during stationary phase (penicillin)
• substance not essential to grow
• dependent on growth conditions
• overproduction possible

1. lactic acid fermentation

• by lactic acid bacteria
• 2 types
• food biotechnology (conservation of milk and vegetables, cheese, yoghurt, pickles, sauerkraut)

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a) HOMOFERMENTATIVE:
• electrons are directly transferred to pyruvic acid
• the only product = lactic acid
• Streptococcus sp., Lactobacillus
• yoghurt, buttermilk, cottage cheese…
• in human muscle!

b) HETEROFERMENTATIVE:
• glycolysis by pentose-phosphate-pathway
• Leuconostoc sp., Lactobacillus
• kefir, sauerkraut

• lactic acid bacteria: gram positive, immotile, rod, spherical shape, aerotolerant anaerobes, high
tolerance of acid, can use lactose as sugar, natural habitats: plants, nasopharynx, intestines, milk,
Streptococcus sp., Lactobacillus sp., Leuconostoc sp.
• used as:
• probiotics (living cells that support human health)
• recombinant proteins (gram positive organisms)

2. alcoholic fermentation
• from pyruvic acid, CO2 is split off
• electrons are transferred to the remaining C2, resulting in ETHANOL
• main producers:
o baker´s yeast (Saccharomyces cerevisiae)
o bacterial EtOH (Zymomonas mobilis)
• maximal EtOH concentration in fermentation: 15%
• higher EtOH concentration: distillation
• EtOH used as beverage, solvent and biofuel

3. antibiotics
• metabolic products of fungi or bacteria, that kill or block the growth of bacteria
• first antibiotics: penicillin (Alexander Fleming)
• TARGETS: many antibiotics act against protein synthesis, but also against cell wall and
cytoplasmatic membrane
• PRODUCTION: tightly controlled process with selected genetically engineered high yield strains
to obtain a high amount of desired product, usually in large fermenters, many purification steps
after fermentation, SANDOZ

• 1. natural antibiotics: no modifications

• 2. biosynthetic antibiotics: modified so that antibiotics increase their efficiency, molecules are
added so that MOs could not produce by itself
• 3. semisynthetic antibiotics: naturally produced structure is chemically or enzymatically altered

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• AMPICILIN: semisynthetic β-lactam antibiotic, destroyed by β-lactamase produced by bacteria,
blocks cross linking in cell wall synthesis (peptides form 1 sugar chain are connected to the other
sugar chain, responsible enzyme for crosslinking peptides in the cell wall is TRANSPEPTIDASE,
penicillin is similar to substrate of transpeptidase – D-ALANINE-ALANINE, D-alanine-alanine
binds to the transpeptidase and blocks its action)

• TETRACYCLINE: produced by soil bacterium – Streptomyces sp., four ring structure, blocks
protein synthesis by preventing binding of tRNA to the mRNA

• KANAMYCIN: aminoglycoside antibiotic, blocks protein biosynthesis that acts on 30S ribosome,
causes misreading of genetic code, inactivated by N-acetyltransferase

• Antimicrobial activity assay/agar diffusion test: antibiotic is applied in different concentrations

to a plate with test organism, zone of inhibition is measured (how resistant the given strain is)

• Minimal inhibitory concentration: antibiotic is applied in different concentrations to a liquid

medium with the test organism, growth is checked

• Resistance: encoded in bacterial DNA or plasmids, antibiotic resistance plasmids, easily spread,
ways (reduces uptake of the antibiotic into the cell or its increased outflux, modification or
inactivation of the antibiotic, alternation of the target site, metabolic bypass)

ORIGIN OF LIFE

• 4 BYA: original of cellular life, anoxygenic phototropic bacteria

• 3 BYA: photosynthesis (oxygen present)
• 2 BYA: modern eukaryotes, unicellular and later multicellular, mammals and humans

Archaea:

• in 1970s, it was discovered that archaea are different from bacteria

• this was done by comparing 16S rRNA sequences
• 16S rRNA sequences have multiple copes within a genome, are conserved and present in all
organisms
• PCR is used to enlarge parts of 16S rRNA in prokaryotes (18S rRNA in eukaryotes)
• obtained sequence is compared to databases full of 16S rRNA sequences
• identical sequence = organism identified
• similar sequence = organism related

= PHYLOGENETIC TREE

Phylogenetic tree:

• last universal common ancestor (LUCA) is divided into bacteria and archaea
• what is interesting is, that eukaryote is to be found on the branch of archaea
• this shows relations in evolution
• the closer the organisms are related, the closer they are on the phylogenetic tree
• branch lengths indicate amount of change since the last branching point (long branch means that
more mutations occurred)

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Phylogeny:

• history of evolution (how life on Earth developed)

• history of group of organisms
• nucleotide sequence data

• align sequences
• distance matrix is calculated from number of sequence
differences
• tree is constructed by adding nodes to join pedigree/lineage that have the least differences

Endosymbiotic theory:

= explanation for the origin of eukaryotic cells by Lynn Margulis in the 1960s = suggests that
eukaryotic cells evolved through a process of endosymbiosis, where one prokaryotic organism
engulfed another and formed a symbiotic relationship. According to the endosymbiotic theory, the
origin of eukaryotic cells involved two major events:

1. respiration:

• prokaryote ingested a bacterium capable of aerobic respiration = host cell formed a symbiotic
relationship with it instead of digesting it
• this then turned into mitochondrion (responsible for respiration in eukaryotic cells)
• Mitochondria have their own DNA and replicate independently within eukaryotic cells, similar to
free-living bacteria. This suggests that mitochondria were once independent organisms that
became integrated into the host cell.

2. photosynthesis:

• prokaryote ingested photosynthetic bacterium like cyanobacterium

• the photosynthetic bacterium formed a symbiotic relationship with the host
• this then turned into chloroplast (responsible for photosynthesis = sunlight into energy)
• Like mitochondria, chloroplasts have their own DNA and can replicate independently within
eukaryotic cells.

Levels of phylogenetic groups:

• domain, phylum, class, family, genus (Escherichia, Homo), species (coli, sapiens), subspecies,
strain
• domain: bacteria, archaea, eukarya

Species in bacteria:

• not easy to define what is species for prokaryotes

• current definition of prokaryotic species:
- collection of strains sharing a high degree in similarity of traits
- most important traits include 70% or greater DNA hybridization and 97% or greater 16S
rRNA gene sequence identity

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 - bacterium is considered a new species, if its 16S rRNA gene sequence differs by more than
3% from any named strain
- bacterium is considered a new genus, if its 16S rRNA gene sequence differs by more than
5% from any named strain
• not all bacteria within a species are identical, because of different pathotypes
• E. coli is not always the same! (different disease causing genes)
• when we have starter culture of 1 bacterium – we achieve high mutation rate
• that is why it is defined for biotechnological processes how many passages are allowed from
starter culture

Archaea vs bacteria:

• prokaryotes
• domains
• cocci, rod, spirulla
• no membrane organelles

• cell wall:
• no peptidoglycan
• some contain pseudopeptidoglycan or S-layer (crystal-like layer of polysaccharide, glycoprotein
or protein), archaea are genetically more similar to eukarya as they contain histones, introns and
several RNAs

• lipids:
• isoprene chains (instead of fatty acids)
• ether linkage (instead of ester linkage)
• L-glycerol (instead of D-glycerol)

• = types: Euryarchaeota, Nanoarchaeota, Korarchaeota,

Crenarchaeota

Euryarchaeota = HALOPHILES:

• live in salty environment (dead sea 30% - 33%)

• die below 10% of NaCl concentration
• some live by aerobic (oxygen) respiration
• Halobacterium makes photosynthesis without chlorophyll
• contains bacteriorhodopsin with cofactor retinal

Euryarchaeota = METHANOGENS:

• strictly anaerobic (no oxygen)

• produce methane from H2 and CO2 or acetate (green-house gas)
• live in marine and freshwater sediments, deep soils, rumen of cows

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Crenarchaeota = HYPERTHERMOPHILES:

• live in hot, volcanic, acidic springs

• optimum temperature range: 75°C – 105°C
• live also in mesothermic (normal) environment or in extreme cold

SUMMARY:

• common ancestor: archaea – eukaryotes

• Loki cut off eukaryotes of the tree of life
• Lokiarchaeota – ASGARD ARCHAEA: close to eukaryotes, contain “eukaryotes only” proteins,
eukaryotes are special types of archaea

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