1. A bioreactor with a kLa of 25 h-1 with active microbes is aerated resulting in a steady oxygen concentration of 1 mg/L. What is the microbial oxygen uptake rate (in mg/L/h) assuming the oxygen saturation concentration is 8 mg/L?

1

• 1. A bioreactor with a kLa of 25 h-1 with active
  microbes is aerated resulting in a steady oxygen
  concentration of 1 mg/L. What is the microbial
  oxygen uptake rate (in mg/L/h) assuming the
  oxygen saturation concentration is 8 mg/L?

2

• 2. The airflow to a Vinegar producing chemostat
  running at steady state was interrupted (at 90
  sec. below) and oxygen data recorded.
• a. What is the kLa of the chemostat in h-1 ?
•  
• b. What was the ethanol (CH3-CH2OH) to acetic
  acid (CH3-COOH) conversion rate of the process
  when it was at steady state?

3

• 4. List (in the box next to the molecule) the
  number of moles of oxygen needed for the complete
  oxidation to CO2 of the following compounds
• CH3-CH2-CH2OH   HOOC-COOH   CH3-CO-CH3  
• 5. List (in the box next to the molecule) the
  number of moles of NAHD that can be generated
  from the complete oxidation to CO2 of the
  following compounds
• Pentose (CH2O)5   CH3-COOH   H2CO3  
• 6. List (in the box next to the molecule) the
  number of moles needed for an anaerobic microbe
  using these substances instead of oxygen as the
  electron acceptor for the complete oxidation to
  CO2 of ethanol (CH3-CH2OH)
• NO3- ? N2   SO42- ? H2S  Fe3 ? Fe2  

4

• 7. Can microbes use the oxygen atom in the H2O
  molecule as an electron acceptor? Give reasons
  for your explanation and an example of the end
  product that would be formed (in the case you
  think it is feasible).

5

• 8. A 10L chemostat is operated with a flowrate of
  0.6 L/h. An equilibrium is established with a
  constant oxygen, concentration, pH, biomass (3
  g/L) and substrate concentration. What is the
  specific growth rate of the microbes in the
  chemostat and what is the biomass productivity R
  (g/L/h) of the chemostat?
• u D F/ V 0.6L/h/ 10L 0.06 h-1
• Productivity R g/L/h can be calculated from X D
• 3g/L 0.06 h-1 0.18 g/L/h

6

• 9. How would you determine the microbial Yield
  coefficient from a batch culture and a chemostat
  culture respectively?

7

• 10. Explain the effect of biomass feedback
  (recycle, retention) on the biomass concentration
  and productivity R of a chemostat. Use a plot of
  biomass (X) and productivity (R) versus the
  dilution rate to illustrate the point.

8

• Effect of biomass feedback (here 3 fold)
• Dotted line no feedback
• Washout occuring early
• 3-fold Feedback? approximately
• 3X? 3R ? 1/3 S
• allows 1/3 reactor size to do same work
• Feedback essential for pollutant removal. Can be
  used 100-fold ? 100-fold smaller treatment plant
• Note same assumed feed concentration (SR)

SR
X
Steady State Concentration
R
S
D
Dcrit
9
Effects of growth constants on steady state
concentrations of biomass and substrate in a
chemostat as a function of dilution rate (x-axis)
Effect of ms
Effect of decrease ks
Effect of increased µmax
Effect of increased Y
10

• 11. Sketch below an example graph of the specific
  growth rate of a microbe (Y-axis) dependent on
  the limiting substrate concentration (X-axis).
  Put a scale (numbers and units) on both axes.
  Point out in your graph (with an arrow) where 3
  of the 4 growth constants can be read from and
  give their values and units as read from your
  example graph.

11
(No Transcript)
12
Substrate limitation of microbial growth
The two curves are described by two
properties The maximum specific growth rate
obtained with no substrate limitation (umax
(h-1)) and the half saturation constant
(Michaelis Menten constat), giving the substrate
concentratation at which half of the maximum u is
reached (ks (g/L)).
Growth- Michaelis Menten model
13
Effect of Maintenance Coefficient (mS) on growth
Rate
The negative specific growth rate (µ) observed in
the absence of substrate (when S 0) (cells are
starving, causing loss of biomass over time) is
the decay rate mSYmax
µ (h-1)
0
S(g/L)
- mSYmax
14
Relationship between oxidation state and electron
equivalents of carbon atoms
OS EE Example 4 0 CO2 3 1 -COOH 2 2 HCOOH, CO,
-CO- 1 3 -CHO 0 4 -CHOH- -1 5 -CH2OH -2 6 -CH
2-, CH3OH -3 7 -CH3 -4 8 CH4

• The electron equivalents (EE) on a carbon atom is
  4 minus the oxidation state (OS)
• EE 4-OS
• Note
• Electron equivalent
• Reducing equivalent
• (degree of reduction)

15
MSE 2011 1) A bioreactor with a kLa of 20 h-1
with active microbes is aerated resulting in a
steady oxygen concentration of 2 mg/L. What is
the microbial oxygen uptake rate (in mg/L/h)
assuming the oxygen saturation concentration is 8
mg/L? OUR 20h-1 (8-2 mg/L) 120 mg/L/h     2)
The airflow to a chemostat running at steady
state DO of 5 mg/L (cS was 8 mg/L) was
temporarily interrupted. The oxygen concentration
decreased steadily by 0.05 mg/L every second.
What is the kLa of the chemostat in h-1 ? kLA
180 mg/L/g / (8-5 mg/L) 60 h-1       3) What
is the maximum possible rate (in mM/h) of lactate
(CH3-CHOH-COOH) oxidation to CO2 by an aerobic
reactor that is limited by an oxygen supply due
to a kLa of 50 h-1 assuming an oxygen saturation
concentration of 8 mg/L? Lac 12 e- ? 1 Lac
reacts with 3 O2 OUR 50 h-1 8mg/L 400
mg/L/h 25 mM/h ? LUR 4.17 mM/h
16
MSE 2011 1) A bioreactor with a kLa of 20 h-1
with active microbes is aerated resulting in a
steady oxygen concentration of 2 mg/L. What is
the microbial oxygen uptake rate (in mg/L/h)
assuming the oxygen saturation concentration is 8
mg/L? OUR 20h-1 (8-2 mg/L) 120 mg/L/h     2)
The airflow to a chemostat running at steady
state DO of 5 mg/L (cS was 8 mg/L) was
temporarily interrupted. The oxygen concentration
decreased steadily by 0.05 mg/L every second.
What is the kLa of the chemostat in h-1 ? kLA
180 mg/L/g / (8-5 mg/L) 60 h-1       3) What
is the maximum possible rate (in mM/h) of lactate
(CH3-CHOH-COOH) oxidation to CO2 by an aerobic
reactor that is limited by an oxygen supply due
to a kLa of 50 h-1 assuming an oxygen saturation
concentration of 8 mg/L? Lac 12 e- ? 1 Lac
reacts with 3 O2 OUR 50 h-1 8mg/L 400
mg/L/h 25 mM/h ? LUR 4.17 mM/h
17
List (in the box next to the molecule) the number
of moles of oxygen needed for the complete
oxidation to CO2 of the following compounds
CH3-CH2-CH2OH   4.5 HOOC-COOH  0.5
CH3-CO-CH3   4     List the four growth
constants with their units. State in one short
sentence what this growth constant means by
referring to its units. Ymax gX/gS umax gX/L/h
/ /gX/L h-1 ms gS/gX/h h-1 kS gS/L  
18
How much NADH can be produced from the complete
oxidation to CO2 of the following
compounds CH3-CHOH-CH2-CH2OH  11
CHOOH  1 benzoate (aromatic ring with a COOH
group attached to one of the carbons  15   Can
microbes use the oxygen atom in the H2O molecule
as an electron acceptor? Give reasons for your
explanation and an example of the end product
that would be formed (in the case you think it is
feasible).           A chemostat is used to
produce microbial biomass for the purpose of
recombinant protein production. Lactate
(CH3-CHOH-COOH) from dairy wastewater is used as
the substrate. The yield coefficient of the
recombinant strain is 0.3 g of cells per g of
lactate degraded. When interrupting the air flow
the oxygen concentration decreased as follows
(time is time in sec after interruption) 0 sec
3 mg/L, 2 sec 2.5 mg/L, 4 sec 2 mg/L, 8 sec
1 mg/L, 12 sec 0.2 mg/L. What is the a) lactate
oxidation rate, b) the biomass productivity (mg
biomass formed/L/h)? OUR 0.25 mg/L/s 900
mg/L/h 28.1 mmol/L/h (MW 32 mg/mmol)/ ? LUR
9.38 mmol/L/h 0.3 g X/ g Lac degraded ? Needed
LUR in mg/L/h ? LUR (312 3 16 6 90mg/mmol)
844.5 mg/L/h ? Productivity 844.5 0.3
253.3 mg/L/h  
19

• A 20L chemostat is operated with a flowrate of
  0.6 L/h. An equilibrium is established with a
  constant oxygen, concentration, pH, biomass
  (2g/L) and substrate concentration. What is the
  specific growth rate of the microbes in the
  chemostat and what is the biomass productivity R
  (g/L/h) of the chemostat?
•  
•   D 0.03 h-1 ? u 0.03 h-1
• X 2 g/L ? R 0.06 gX/L/h
• In the absence of oxygen, many bacteria can use
  nitrate (NO3-) as electron acceptor and produce
  N2 as the endproduct (nitrate respiration or
  denitrification). What rate of nitrate reduction
  to N2 would you expect of a reactor that was
  switched from aerobic (aerated) conditions to
  nitrate reducing conditions, if the aerobic
  reactor had an oxygen uptake rate of 80 mg/L/h?
•  
•  NO3- ? N2 requires 5 e- while O2 ? H2O
  requires 4 e-
• NUR 4/5 OUR (molar)
• OUR 80mg/L/h / 32 mg/mmol 2.5 mmol/L/h
  ? NUR 2 mmol/L/h
•  
•  
• Contrast batch culture against chemostat culture
  by pointing out advantages and limitations.
•  
•  Chem higher productivity, easier automation,
  ideal for study
• Chem- not for secondary metabolites, prone to
  cont from outside and backmutations
•  
•  

20
      How can you calculate the productivity of a
chemostat? Give 3 examples of how the
productivity of a chemostat can be approximately
doubled by the operator and one statement for
each example how this works. R (gX/L/h) D
(h-1) X (g/L) Can be increased by operator by
increasing either D or X D Double flowrate  X
Double SR X Retain bacteria by recycle or filter
to twice the concentration
21
Growth- Simplified Scheme of Energy preservation
as ATP
Biological growth requires ATP as the energy
source (energy rich phosphate-phosphate
bond). ATP is generated mostly during Respiration
(Dissimilation) ATP then drives the biomass
synthesis (Assimilation) How is it generated
? How much is generated ?
22
Energy preservation as ATP

• Four steps for aerobic ATP generation from
  glucose
• Glycolysis sugar ? acetate (C2))
• TCA cycle acetate ? CO2 4 NADH
• NADH O2 ? NAD proton gradient
• Proton gradient runs a nano-scale turbine
  called ATP synthase

23
Growth- Overview of Energy Metabolism simplifying
FAD and ATP genration in TCA
CO2
glucose
glucolysis
2 acetate
TCA cycle
Cell
1 ATP ? 3 H
2 NADH
8 NAD
ATP synthase
8 NADH
ETC
Overall 36 ATP (2) allowing growth
O2
2 NADH
1NADH ? 9 H
24
Growth- Simplified Scheme of Energy preservation
as ATP
Important Quantities ATP-synthase 3H ? 1
ATP ETC 1 NADH ? 33 9 H 2 NADH reduce 1
O2 glycolysis 1 glucose ? 12 NADH 1 glucose ?
129 108 H 36 ATP 2 ATP generated from
glycolysis via substrate level phosphorylation
38 ATP
?1NADH ? 3 ATP
25
Energy Source for Growth

• Electron flow
• is critical for the understanding of microbial
  product formation
• allows to understand fermentations
• the rate of electron flow determines the
  metabolic activity
• Which direction? ? Thermodynamics
• How powerful ?? Thermodynamics
• How rapid ? ? Kinetics
• How many ? ? Stoichiometry, mass balance,
  fermentation balance

26
Growth- Simplified Scheme of Energy preservation
as ATP
How does ATP synthase work? A mechanical turbine
that generates a energy rich phopspate bond
driven by a proton gradient across the cell
membrane See animated clip.
27
Energy Source for Growth

• Microbes catalyse redox reactions (electron
  transfer reactions)
• A redox reaction oxidises one compound while
  reducing another compound
• The electron flow represents the energy source
  for growth
• An energy source must have an electron donor and
  electron acceptor

oxidation
Electron donor (Reductand)
Electron Carrier
reduction
Electron acceptor (Oxidant)
Electron flow (arrows) electron donor to
electron acceptor
28
Energy Source for Growth

• Electron flow
• Which direction? ? Thermodynamics
• How powerful ?? Thermodynamics
• How rapid ? ? Kinetics
• How many ? ? Stoichiometry, mass balance,
  fermentation balance

oxidation
Electron donor (Reductand)
Electron Carrier
reduction
Electron acceptor (Oxidant)
Electron flow (arrows) electron donor to
electron acceptor
29
Energy Source for Growth

• What are electron carriers?
• A redox couple that mediates between donor and
  acceptor
• A redox couple consists of the oxidised and the
  reduced form (e.g. NADH and NAD)
• acts also as reducing equivalents buffer
• What are suitable electron donors and acceptors?

oxidation
Electron donor (Reductand)
Electron Carrier
reduction
Electron acceptor (Oxidant)
Electron flow (arrows) electron donor to
electron acceptor
30
Growth- Simplified Scheme of Energy preservation
as ATP
What do electron carriers look like?
31
Working principle of electron carriers

• What are electron carriers?
• A redox couple that mediates between donor and
  acceptor
• A redox couple consists of the oxidised and the
  reduced form (e.g. NADH and NAD)
• electron buffer
• What are suitable electron donors and acceptors?

OH
O
OH
O
Electron carriers exist as a couple
32
Working principle of electron carriers

• What are electron carriers?
• A redox couple that mediates between donor and
  acceptor
• A redox couple consists of the oxidised and the
  reduced form (e.g. NADH and NAD)
• electron buffer
• What are suitable electron donors and acceptors?

OH
O
OH
O
Electron carriers exist as a couple
33
Working principle of electron carriers (EC)

• What is the most important difference between the
  two forms?
• Different number of double bonds
• OH instead of O

OH
O
OH
O
Quinone and hydroquinone as central pieces of
Ubiquinone
34
Working principle of electron carriers (EC)

• Which form carries electrons?
• The reduced form!
• Which is the reduced form?
• The oxidation states will tell!
• Which carbon atoms changed their oxidation state?

OH
O
OH
O
Quinone and hydroquinone as central pieces of
Ubiquinone
35
Working principle of electron carriers (EC)

• Which carbon atoms changed their oxidation state?
• All carbons that have just one H bonded maintain
  OS of -1
• The top and bottom C have changed their OS.

OH
H
H
O
H
H
H
H
OH
H
H
O
Quinone and hydroquinone as central pieces of
Ubiquinone
36
Working principle of electron carriers (EC)

• Which carbon atoms changed their oxidation state?
• All carbons that have just one H bonded maintain
  OS of -1
• The top and bottom C have changed their OS.
• The reduced form carries two more electrons than
  the oxidised form
• Where are they?

OH
1
H
H
O
H
H
2
1
H
H
OH
H
H
2
O
Quinone and hydroquinone as central pieces of
Ubiquinone
37
Working principle of electron carriers (EC)

• Which carbon atoms changed their oxidation state?
• All carbons that have just one H bonded maintain
  OS of -1
• The top and bottom C have changed their OS.
• The reduced form carries two more electrons than
  the oxidised form
• Where are they?

OH
H
H
O
H
H
1
H
H
OH
H
H
2
O
Quinone and hydroquinone as central pieces of
Ubiquinone
38
Working principle of electron carriers (EC)

• How many electrons are carried ?
• 2
• What else is carried?
• a proton
• Together the electron and the proton make one H
• The reduced electron carrier can also be called a
  hydrogen carrier?
• Hydrogenation adding hydrogen or electrons to
  another compound reducing the compound

OH
H
H
O
H
H
1
H
H
OH
H
H
2
O
Quinone and hydroquinone as central pieces of
Ubiquinone
39
Working principle of electron carriers (EC)

• What can a reduced EC do?
• Does a cell also need oxidised EC?

OH
H
H
O
H
H
1
H
H
OH
H
H
2
O
Quinone and hydroquinone as central pieces of
Ubiquinone
40
Working principle of electron carriers (EC)
H
-1

• The electrons in NADH as the most importanT
  electron carrier can also be visualised
• as N is more electronegative than C it is
  allocated the electrons of C-N bonds (similar to
  oxygen)

R
H
1
H
H
N
H
H
-2
R
R
H
0
H
H
N
R
NADH/NAD as electron carrier
41
Main advantage of reducing power (NADH)

• aerobic conditions, NADH ? ATP generation
• NADH H 0.5O2 3 ADP 3Pi ? NAD 3 ATP 4 H2O
• Respiration balance combination of ETC and ATP
  synthase reaction
• How useful is NADH without O2 ?

42
Consequences of O2 depletion on cells

• Consequences of O2 depletion
• No ATP generation
• NAHD accumulates and NAD is depleted
• TCA cycle (requiring NAD) cant run
• glucose uptake stops
• NADH (or NADPH) can also be used for anabolism
  (assimilation)
• but in addition to reducing power also ATP is
  needed for assimilation
• Without O2 NADH is a problem rather than
  advantage
• Anaerobic organisms have developed special
  metobolic pathways to re-oxidise NADH
  (fermentations and anerobic respirations)

43
Energy Metabolism Scheme simplifying FAD and ATP
genration in TCA
CO2
glucose
glucolysis
2 acetate
TCA cycle
Cell
1 ATP ? 3 H
2 NADH
8 NAD
ATP synthase
8 NADH
ETC
Overall 36 ATP (2) allowing growth
O2
2 NADH
1NADH ? 9 H
44
Electron flow in fermentations.

• Anaerobic fermentations (strict sense) make use
  of internal organic electron acceptors .
• The electron flow in anaerobic fermentations can
  be easily demonstrated by documenting the changes
  in carbon numbers and electron numbers.
• For example glucose (CH2O)6 contains 6 carbons
  with an oxidation state of zero (4
  electrons/carbon).
• Glucose can be presented as 6 C, 24 e-

45
Lactic acid fermentation .

• Anaerobic fermentations (strict sense) make use
  of internal organic electron acceptors .
• The electron flow in anaerobic fermentations can
  be easily demonstrated by documenting the changes
  in carbon numbers and electron numbers.
• For example glucose (CH2O)6 contains 6 carbons
  with an oxidation state of zero (4
  electrons/carbon).
• Glucose can be presented as 6 C, 24 e-

46
Lactic Fermentation - Electron and carbon flow -
ATP
ATP
LDH
LDH
lactate
glucose (CH2O)6
2 red. equiv.
pyruvate (CH3-CO-COOH)
hydroxy propanoate lactate (CH3-CHOH-COOH)
LDH Lactate dehydrogenase enzyme
47
Notes on origin of enzyme namesWith 2 electrons
also 2 protons are transferred? electron
transfer hydrogen transferRemove e-/H2
Dehydrogenation oxidationAdd e-/H2
Hydrogenation reduction Pyruvate 2e- ?
LactatePossible names for the enzyme catalysing
the equilibrium (forward and backward
reaction)Lactate dehydrogenaseLactate
oxidasePyruvate hydrogenasePyruvate reductase
48
Quizz Glucose(6 carbons) is fermented to 2
lactate(CH3-CHOH-COOH) 123 If instead ethanol
(CH3-CH2OH) 122 is the end product, how many can
be formed?Carbon balance would suggest 3 (2
carbons)!Electron balance suggests 2 (12
electrons) ? Electrons are relevant, not
carbon.If electrons are balanced any extra
carbon must be in the form of CO2.
49
Ethanolic Fermentation - Electron and carbon flow
-
glucose
ATP
ATP
PDC
PDC
EDH
EDH
glucose
ethanol
2 red. equiv.
pyruvate
Key enzymes PDC pyruvate decarboxylase EDH
Ethanol dehydrogenase
acetaldehyde
ethanol
50
The Entner Doudoroff (KDPG) pathway of ethanolic
fermentation
Orgainism Zymonas mobilis
glucose
gluconate
GAP
pyruvate
ATP
CO2.
acetaldehyde
ethanol
51
Application of Lactic Fermentation - Silage -

• Silage Lactic acid fermentation of fodder
  material
• Better preservation of food energy value than by
  drying (hay)
• Process
• Rapid filling of tank (silo)silo with shredded
  material
• Additves (germination inhibitors, sugars, pH
  controlers)
• Packing densely and compressing
• Sealing air-tight
• Avoid contaminatin with decaying material
  (proteolytic anaerobes such as Clostridia

52
Silage does not necessarily need a tank Examples
of silage in Australia
53
Overview of Energy Metabolismsimplifying FAD and
ATP genration in TCA
glucose
TCA cycle
glucolysis
Cell
ATP synthase
glucose ? 12 NADH 2 ATP
3H ? 1ATP
Keywords to look up Electron carriers Proton
gradient electron motive force Hydrogenation
Reduction Dehydrogenation Oxidatioin
ETC
38 ATP
NADH ? 9 H
54
ConclusionIn the absence of O2 fermentations
can be carried out that transfer electrons to
internal (synthesised) electron acceptors instead
of oxygen.Useful bioproducts can be
obtainedEthanol, organic acids, H2
55
Lec 5 Overview Microbial metabolism without O2

• Microbial growth is driven by the energy released
  from the transfer of electrons from donor
  (reductant, typically organic compounds) to
  acceptor (oxidant, typically oxygen.
• The transfer occurs via mediators (electron
  carriers)
• In the absence of oxygen microbes can ferment
  sugars by using internal organic mediators (e.g.
  puruvate, or acetaldehyde) resulting in
  fermentation products such as ethanol and lactic
  acid (hydroxy propnanoic acid)
• The number of electrons available for reductions
  (reducing equivalents) on organic substances
  (including mediators) can be derived from the
  oxidation states of the carbons

56
Ethanolic Fermentation - Electron and carbon flow
-
OH H C H H C H H
O.S. -1 ? 5 electrons
O.S. -3 ? 7 electrons

• Energy conserved
• 2 ATP from glycolysis (PGK, PK)

• Key enzymes
• Pyruvate Decarboxylase,
• Ethanol Dehydrogenase
• (could also be called ethanol oxidase or
  acetaldehyde reductase)

57
The Entner Doudoroff (KDPG) pathway of ethanolic
fermentation
Organism Zymonas mobilis (not examined)
glucose
gluconate
GAP
pyruvate
ATP
CO2.
acetaldehyde
ethanol
58
Special features of Entner Doudoroff pathway

• 1 NADH, 1 NADPH

• Only 1 ATP (less biomass as byproduct)

• Only one pyruvate through GAP (bottleneck) ?
  faster?

Special features of Zymomoanas

• Higher glucose tolerance

• Higher product yield (less ATP ? less biomass)
  (100 g ethanol / 250 g glucose) 78 molar conv.
  eff

• Not higher ethanol tolerance

59
Special features of Entner Doudoroff pathway (not
examined)

• 1 NADH, 1 NADPH

• Only 1 ATP (less biomass as byproduct)

• Only one pyruvate through GAP (bottleneck) ?
  faster?

Special features of Zymomoanas

• Higher glucose tolerance

• Higher product yield (less ATP ? less biomass)
  (100 g ethanol / 250 g glucose) 78 molar conv.
  eff

• Not higher ethanol tolerance

60
Bio-ethanol from sugar cane as fuel (Brasil)

• Distillation costs more energy than ethanol fuel
  value
• Separation costs higher than fermentation costs

• Research (1990s)
• Thermophilic strains (Clostridium using
  cellulose)
• Finding more ethanol resistant strains
• Controversial topic
• Bioethanol from sugar (first generation
  bio-ethanol) has
• ethical problems.
• Current research
• Bio-ethanol from cellulosic waste (straw, wood,
  paper)
• Requires enzymes. (e.g. Simultaneous
  saccharification/ fermentation)

61
Lactic Fermentation - Occurrence -

• If plant or animal material containing sugars and
  complex nitrogen sources is left in the absence
  of oxygen ? lactic acid bacteria take over ?
• Selective enrichment
• Natural fermentation (since prehistoric times)
• Why do lactic acid bacteria take over sugar
  conversion on rich media?
• Simple metabolism ? fast degradation
• 2) Amino acids are not synthesized but taken up
  from the medium ? faster growth
• 3) Strains are existing on substrate (e.g. milk,
  vegetables)
• 4) O2 tolerance of strains
• 5) Production of inhibitory acid (ph lt5)
• Examples Milk, whole meal flour, vegetables,

62
Lactic Fermentation - Organisms -

• Lactic acid bacteria (Lactobateriacease)
• gram positive
• non motile
• obligate anaerobics
• no spores
• aerotolerant
• no cytochromes and catalase
• fermentation of lactose
• no growth on minimal glucose media
• requirement of nutritional supplements
  (vitamins, amino acids, etc.)
• when supplied with porphyrins ? they form
  cytochromes !?! (indicating that they were
  originally aerobic organisms that have lost the
  capacity of respiration, metabolic cripples)

63
Homolactic Fermentation - Electron and carbon
flow -
ATP
ATP
LDH
LDH
lactate
glucose
LDH lactate dehydrogenase
2 red. equiv.
pyruvate
lactate
64
Homo-lactic Fermentation - Electron and carbon
flow -
O CH C H C H H C H H
O.S. 3 ? 1 electron
O.S. 0 ? 4 electrons
O.S. -3 ? 7 electrons
Strategy
1) Aerotolerant ? can ferment with strict
anaerobes are still inhibited by oxygen
2) Simple quick metabolism and usage of
carbohydrates
3) Production of acid, inhibiting competitors
65

• Significance
• Why do lactic acid bacteria not spoil food but
  preserve it?
• Only ferment sugars (24 e-) to lactate (2 12 e-)
  ? nutritional value not significantly altered
• Dont degrade proteins
• Dont degrade fats
• Acidity suppresses growth of food spoiling
  organisms (eg. Clostridia)
• enhances nutritional value of organic material
  (example sauerkraut, Vit. C, scurvy)
• Complex flavour development (diacetyl)
• Examples
• Yogurt, sauerkraut, buttermilk, soy sauce, sour
  cream, cheese, pickled vegetables,
• technical lactic acid for the production of
  bio-plastic (hydroxy acids allow chain linkages
  via ester bonds between hydroxy and carboxy
  group).

66
Heterolactic Fermentation Phosphoketolase pathway
glucose
ribose
2 red. equiv.
pyruvate
ATP
lactate
ethanol
acetate
CO2.
Phosphoketolase pathway combination of
Pentosephosphate cycle and FBP pathway
67
Heterolactic Fermentation Phosphoketolase pathway
glucose
ribose
2 red. equiv.
pyruvate
ATP
lactate
ethanol
acetate
CO2.
Presence of oxygen ? lactate, acetate and CO2
production ? 1 additional ATP from acetokinase.
No ETP
68
Heterolactic Fermentation
Organisms E.g. Leuconostoc spp. Lactobacillus
brevis

• Strategy
• Use of parts of the pentose phosphate cycle
  which is designed for synthesis of pentose (DNA,
  RNA). ?

• Aerotolerant, simple pathway, quick metabolism,
  suited for substrate saturation.

Application Sourdough bread, Silage, Kefir,
Sauerkraut, Gauda cheese (eyes)
In the presence of oxygen, reducing equivalents
from glucose oxidation are transferred to oxygen,
allowing the gain of an additional ATP via
acetate excretion
Key enzymes of FBP pathway missing (Aldolase,
Triosephosphate isomerase).
69
Application of Lactic Fermentation

• Silage Lactic acid fermentation of fodder
  material
• Process
• 1) partial drying of fodder
• 2) shredding
• 3) Rapid filling of silo (1 or 2 days)
• 4) packing as densely as possible
• 5) Compressing
• 6) Sealing airtight
• 7) Additives (germination inhibitors, sugars,
  organic acids)
• 8) Avoid contamination with decaying fodder
  (Clostridia, proteolytic bacteria)
• Nutrient loss
• drying of fodder ? hay (25),
• ensilaging (10) (2ATP out of 38)

70
Applications of Lactic Fermentation
Sauerkraut
In principle identical to silage with following
modifications
1) White cabbage as the only plant material
2) Cabbage mixed with NaCl (2 2.5)
3) Capacity of vessels (concrete, wood) up to 100
tons
4) Incubation (18oC to 20oC) for 4 weeks
5) Recirculation of brine by pumping for process
monitoring (acids)
6) About 1.5 lactic acid produced
7) Sterilisation of product to have cooked
sauerkraut (German). Raw (fresh sauerkraut used
in salads)
8) Problem 1 to 15 tons of highly polluted
effluent per ton of cabbage
71
Applications of Lactic Fermentation
Sauerkraut

• Similar to silage with following modifications
• White cabbage as the only plant material
• 2) Cabbage mixed with NaCl (2 2.5)
• 3) Capacity of vessels (concrete, wood) up to 100
  tons
• 4) Incubation (18oC to 20oC) for 4 weeks
• 5) Recirculation of brine by pumping for process
  monitoring (acids)
• 6) About 1.5 lactic acid produced
• 7) Sterilisation of product to have cooked
  sauerkraut (German). Raw (fresh sauerkraut used
  in salads)
• 8) Problem 1 to 15 tons of highly polluted
  effluent per ton of cabbage

Brine Recycle
72
Applications of Lactic Fermentation
Brine Recycle
73
Applications of Lactic Fermentation
Olives
1) Black (ripe) or green (unripe) olives
2) Pretreatment with 1.5 NaOH saline (reducing
bitterness)
3) Washing
4) Place fruit (still alcaline) in brime of 10
NaCl 3 lactic acid (to neutralise pH)
5) Sugar addition to accelerate fermentation
(Lactobacillus plantarum)
6) Incubate for several months until lactic acid
gt0.5
7) Wooden barrels or plastic tanks
74
Pickled Gherkins
1. Cover gherkins in 3 salt brine (NaCl)
2. Add spices, herbs, dill
3. Irradiate surface (UV) and close vessel
4. After 3 6 weeks 3 lactic acid is produced
5. Fermentation pattern like silage
75
Applications of Lactic Fermentation
Technical lactic acid
Use Leather Textile and Pharmaceutical
Industry
Bioplastics (Polylactic acid, biodegradable)
Food acid (flavourless, non volatile) e.g. in
sausages
Product yield 900 g per g of sugar
Substrate whey, cornsteep liquor, malt
extract, ideally sugars (15 cane or beets)
Strains Lactobacillus bulgaricus, Lactobacillus
delbrueckii
Duration 5 days batch culture
76
Applications of Lactic Fermentation
Sourdough bread
Biological raising agent (homo- and heterolactic
fermentation)
CO2 produced from heterolactic bacteria
Necessary for rye bread to increase digestibility
Health bread (lipid, proteins unchanged, vitamins
produced)
Pre-acidified (stomach friendly)
Complex flavour development
Increased shelf life
77
Cheese Production
Milk
Homogenise
Add starter culture (S. cremoris, S. lactis, L.
bulgaricus, S. thermophilus
Pasteurise
Add Rennet
Curdling Stirring Settling
Yougurt (430)
Scolding Cooling Washing Salting
Heat treatment (600) Kneading
Whey
Quark Fromage frais (acidic paste)
Whey
Cottage cheese (granular)
Pressuring Maturing
Proteolytic enzyme Coagulating
Heated stirring
Brie Edamer
Cheddar
78
Propanoate Formation From Lactate

• Acryloyl pathway (Clostridium propionicum)
• The 4 reducing equivalents from lactate oxidation
  to acetate
• are merely dumped onto two further moles of
  lactate
• (dismutation, disproportionation)

LDH
PrDH
PDH
ATP
Enzymes Lactate DH, Pyruvate DH, Propionate DH
(PrDH)
79
Propanoate Formation From Lactate

1. Acryloyl pathway (Clostridium propionicum)

Energetic benefit? The excretion of acetate
gains 1 ATP (acetate kniase), Thus 1/3
ATP/lactate metabolised.
LDH
PrDH
PDH
ATP
How to generate ATP from acetate
excretion Phosphate Acetyl transferase AcetateCo
A Pi ? Acetyl-P CoA Acetokinase Acetyl-P
ADP ? Acetate ATP
80
Propanoate Formation From Lactate
2. Methyl-Malonyl-Pathway (Propionibacteria) 2
reducing equivalents from lactate oxidation
(exactly PDH and ferredoxin as e- carrier) are
transferred via electron transport
phosphorylation to fumarate (fumarate
respiration) resulting in one extra ATP (2/3
ATP/lactate metabolised). Reverse TCA
cycle. Fumarate reduction is an example of
anaerobic respiration Homoacetogenesis is another
example
81
Propanoate Formation From Lactate
2. Methyl-Malonyl-Pathway (Propionibacteria)
lactate
LDH
propionate
PDH
ATP
succinate
fumarate (malate)
ATP
OAA
pyruvate
82
Propionic Fermentation of Glucose
83
Propionic Fermentation of Glucose
84
Propionic Fermentation of Glucose
85
Butyric Fermentation
86
Acetone Butanol fermentation
87
Homoacetogenesis
The homoacetogenesis starts like the butyric acid
fermentation 1) Use of the fructose
bisphosphate pathway (FBP) leading to 2 puruvate
and 2 NADH. 2) Oxidative decarboxylation of
pyruvate to acetyl-CoA, hydrogen gas and CO2. 3)
In contrast to the butyric fermentation no
acetoacetyl-CoA is formed. Instead two acetyl-CoA
are intermediate products.
88
Homoacetogenesis
89

• Specific growth rate u in chemostat culture
• Get the D from F/V
• Du

90

• E- acceptor from NADH in fermentations
• For example acetaldehyde in ethanolic ferm

91

• Effect of growth constants on productivity R in a
  chemostat
• R depends on X and D
• Increased umax allows higher D
• Increased Ymax gives higher X
• Ms not much diff

92

• OUR is 64 mg/L/h 2 mmol O2/L/h
• What is the acetone (CH3-CO-CH3) oxidation rate
  to CO2.
• 16 e- means that 4 O2 accept all el from acetone
• Acetone ox rate is 0.5 mmol/L/h

93

• OUR is 64 mg/L/h 2 mmol O2/L/h
• What is the nitrate NO3- to N2 reduction rate
• NUR 2 mmol/L/h 4/5