1. a bioreactor with a kla of 25 h-1 with active microbes is aerated resulting in a steady oxygen...
DESCRIPTION
a. What is the kLa of the chemostat in h-1 ? 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?TRANSCRIPT
![Page 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/1.jpg)
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?
![Page 2: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/2.jpg)
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?
![Page 3: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/3.jpg)
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 SO4
2- H2S Fe3+ Fe2+
![Page 4: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/4.jpg)
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).
![Page 5: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/5.jpg)
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
![Page 6: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/6.jpg)
9. How would you determine the microbial Yield coefficient from a batch culture and a chemostat culture respectively?
![Page 7: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/7.jpg)
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.
![Page 8: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/8.jpg)
X
S
D
Ste
ady
Sta
te C
once
ntra
tion
Effect of biomass feedback (here 3 fold):Dotted line no feedback:•Washout occuring early
•3-fold Feedback approximately:•3*X 3*R 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)
R
Dcrit
SR
And 88
![Page 9: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/9.jpg)
Effects of growth constants on steady state concentrationsof biomass and substrate in a chemostat as a function of dilution rate (x-axis)
Effect of ms Effect of decrease ks
Effect of increased Y Effect of increased μmax
And 99
![Page 10: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/10.jpg)
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.
![Page 11: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/11.jpg)
![Page 12: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/12.jpg)
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)).
Substrate limitation of microbial growth
µ(h-1)
Substrate (g/L)
substratelimitation
kS
µmax(h-1)
Growth- Michaelis Menten modelAnd 1212
![Page 13: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/13.jpg)
µ(h-1)
S(g/L)
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 mS*Ymax
- mS*Ymax
0
Effect of Maintenance Coefficient (mS) on growth Rate
And 1313
![Page 14: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/14.jpg)
Relationship between oxidation state and electron equivalents of carbon atoms
• 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)
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 -CH2-, CH3OH-3 7 -CH3-4 8 CH4
![Page 15: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/15.jpg)
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 k La 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 O2OUR = 50 h-1* 8mg/L = 400 mg/L/h = 25 mM/h LUR = 4.17 mM/h
![Page 16: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/16.jpg)
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 k La 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 O2OUR = 50 h-1* 8mg/L = 400 mg/L/h = 25 mM/h LUR = 4.17 mM/h
![Page 17: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/17.jpg)
List (in the box next to the molecule) the number of moles of oxygen needed for the complete oxidation to CO 2 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/gSumax gX/L/h / /gX/L = h-1ms gS/gX/h = h-1kS = gS/L
![Page 18: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/18.jpg)
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 (3*12 + 3* 16 +6= 90mg/mmol)= 844.5 mg/L/h
Productivity = 844.5 * 0.3 = 253.3 mg/L/h
![Page 19: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/19.jpg)
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 studyChem-: not for secondary metabolites, prone to cont from outside and backmutations 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.
![Page 20: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/20.jpg)
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 XD: Double flowrate X: Double SRX: Retain bacteria by recycle or filter to twice the concentration
![Page 21: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/21.jpg)
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 ?
Growth- Simplified Scheme of Energy preservation as ATP
![Page 22: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/22.jpg)
Four steps for aerobic ATP generation from glucose:
1) Glycolysis : sugar acetate (C2))
2) TCA cycle: acetate CO2 + 4 NADH
3) NADH + O2 NAD + proton gradient
4) Proton gradient runs a nano-scale “turbine” called ATP
synthase
Energy preservation as ATP
![Page 23: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/23.jpg)
glucose
TCA cycle
ETC
1 ATP 3 H+
glucolysis
8 NADH
1NADH 9 H+
Overall:36 ATP (+2)
allowing growth
Cell
O2
Growth- Overview of Energy Metabolismsimplifying FAD and ATP genration in TCA
CO2
2 NADH2
acet
ate
ATPsynthase
2 NADH
8 NAD+
![Page 24: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/24.jpg)
Important Quantities:
ATP-synthase: 3H+ 1 ATP
ETC: 1 NADH 3*3 = 9 H+
2 NADH reduce 1 O2
glycolysis: 1 glucose 12 NADH
1 glucose 12*9 = 108 H+ = 36 ATP
+ 2 ATP generated from glycolysis via substrate level
phosphorylation = 38 ATP
Growth- Simplified Scheme of Energy preservation as ATP
1NADH 3 ATP
![Page 25: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/25.jpg)
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
![Page 26: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/26.jpg)
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.
Growth- Simplified Scheme of Energy preservation as ATP
![Page 27: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/27.jpg)
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
Electron donor(Reductand)
oxidation
reduction
ElectronCarrier
Electron acceptor(Oxidant)
Electron flow (arrows) electron donor to electron acceptor
![Page 28: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/28.jpg)
Energy Source for Growth
Electron flow:
•Which direction? Thermodynamics
•How powerful ? Thermodynamics
•How rapid ? Kinetics
•How many ? Stoichiometry, mass balance, fermentation balance
Electron donor(Reductand)
oxidation
reduction
ElectronCarrier
Electron acceptor(Oxidant)
Electron flow (arrows) electron donor to electron acceptor
![Page 29: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/29.jpg)
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?
Electron donor(Reductand)
oxidation
reduction
ElectronCarrier
Electron acceptor(Oxidant)
Electron flow (arrows) electron donor to electron acceptor
![Page 30: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/30.jpg)
What do electron carriers look like?
Growth- Simplified Scheme of Energy preservation as ATP
![Page 31: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/31.jpg)
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?Electron carriers exist asa couple
OH
OH
O
O
![Page 32: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/32.jpg)
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?Electron carriers exist asa couple
OH
OH
O
O
![Page 33: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/33.jpg)
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
Quinone and hydroquinoneas central pieces of Ubiquinone
OH
OH
O
O
![Page 34: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/34.jpg)
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?
Quinone and hydroquinoneas central pieces of Ubiquinone
OH
OH
O
O
![Page 35: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/35.jpg)
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.
Quinone and hydroquinoneas central pieces of Ubiquinone
OH
OH
O
O
H
H
H
H
H
HH
H
![Page 36: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/36.jpg)
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?Quinone and hydroquinoneas central pieces of Ubiquinone
OH
OH
O
O
H
H
H
H
H
HH
H
+2
+2
+1
+1
![Page 37: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/37.jpg)
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?Quinone and hydroquinoneas central pieces of Ubiquinone
OH
OH
O
O
H
H
H
H
H
HH
H
+2
+1
![Page 38: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/38.jpg)
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 compoundQuinone and hydroquinone
as central pieces of Ubiquinone
OH
OH
O
O
H
H
H
H
H
HH
H
+2
+1
![Page 39: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/39.jpg)
Working principle of electron carriers (EC)
• What can a reduced EC do?• Does a cell also need
oxidised EC?
Quinone and hydroquinoneas central pieces of Ubiquinone
OH
OH
O
O
H
H
H
H
H
HH
H
+2
+1
![Page 40: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/40.jpg)
Working principle of electron carriers (EC)
• 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)
NADH/NAD+ as electroncarrier
H
RH
R
H
R
H
H
R
HH
H
-2
-1
H
N
N
+1
0
![Page 41: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/41.jpg)
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 ?
![Page 42: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/42.jpg)
Consequences of O2 depletion on cells
Consequences of O2 depletion: • No ATP generation• NAHD accumulates and NAD+ is depleted• TCA cycle (requiring NAD+) can’t 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)
![Page 43: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/43.jpg)
glucose
TCA cycle
ETC
1 ATP 3 H+
glucolysis
8 NADH
1NADH 9 H+
Overall:36 ATP (+2)
allowing growth
Cell
O2
Energy Metabolism Schemesimplifying FAD and ATP genration in TCA
CO2
2 NADH2
acet
ate
ATPsynthase
2 NADH
8 NAD+
![Page 44: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/44.jpg)
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-
![Page 45: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/45.jpg)
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-
![Page 46: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/46.jpg)
103 20 103
123 123
20LDH LDH
lactate
ATPATP
246
24620
103123
= glucose (CH2O)6
= 2 red. equiv.
= pyruvate (CH3-CO-COOH)
= hydroxy propanoate =lactate (CH3-CHOH-COOH)
Lactic Fermentation- Electron and carbon flow -
LDH = Lactate dehydrogenase enzyme
![Page 47: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/47.jpg)
Notes on origin of enzyme names
With 2 electrons also 2 protons are transferred electron transfer= hydrogen transfer:
Remove 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
![Page 48: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/48.jpg)
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.
![Page 49: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/49.jpg)
103 20 103
102 102
122 122
20
0101PDCPDC
EDH EDH
ethanol
ATPATP
246 glucose
246
20
103
102
122
= glucose
= 2 red. equiv.
= pyruvate
= acetaldehyde
= ethanol
Ethanolic Fermentation- Electron and carbon flow -
Key enzymes:PDC = pyruvate decarboxylaseEDH = Ethanol dehydrogenase
![Page 50: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/50.jpg)
226
20
102
103 103
122
12320
0101
ATP
246 246
01
103
102
122
= glucose
= CO2.
= pyruvate
= acetaldehyde
= ethanol
102
122
242 = gluconate
123 = GAP
The Entner Doudoroff (KDPG) pathway of ethanolic fermentationOrgainism: Zymonas mobilis
![Page 51: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/51.jpg)
Application of Lactic Fermentation- Silage -
Silage: Lactic acid fermentation of fodder materialBetter preservation of food energy value than by drying (hay)
Process:
1) Rapid filling of tank (silo)silo with shredded material2) Additves (germination inhibitors, sugars, pH controlers)3) Packing densely and compressing4) Sealing air-tight5) Avoid contaminatin with decaying material (proteolytic
anaerobes such as Clostridia
![Page 52: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/52.jpg)
Silage does not necessarily need a tank: Examples of silage in Australia
![Page 53: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/53.jpg)
Overview of Energy Metabolismsimplifying FAD and ATP genration in TCA
glucose
TCA cycle
ETC
ATPsynthase3H+ 1ATP
glucolysisglucose 12 NADH + 2 ATP
NADH 9 H+
38 ATP
Keywords to look up:Electron carriersProton gradientelectron motive forceHydrogenation = ReductionDehydrogenation = Oxidatioin
Cell
![Page 54: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/54.jpg)
Conclusion:
In 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
![Page 55: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/55.jpg)
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
![Page 56: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/56.jpg)
OH
H C H
H C H
H
Ethanolic Fermentation- Electron and carbon flow -
• Energy conserved: 2 ATP from glycolysis (PGK, PK)• Key enzymes: •Pyruvate Decarboxylase, •Ethanol Dehydrogenase
(could also be called ethanol oxidase or acetaldehyde reductase)
O.S.: -1 → 5 electrons
O.S.: -3 → 7 electrons
![Page 57: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/57.jpg)
226
20
102
103 103
122
12320
0101
ATP
246 246
01
103
102
122
= glucose
= CO2.
= pyruvate
= acetaldehyde
= ethanol
102
122
242 = gluconate
123 = GAP
The Entner Doudoroff (KDPG) pathway of ethanolic fermentationOrganism: Zymonas mobilis(not examined)
![Page 58: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/58.jpg)
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
![Page 59: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/59.jpg)
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
![Page 60: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/60.jpg)
Bio-ethanol from sugar cane as fuel (Brasil) • Distillation costs more energy than ethanol fuel value• Separation costs higher than fermentation costs
Research (1990’s)• Thermophilic strains (Clostridium using cellulose)• Finding more ethanol resistant strains
Controversial topic: Bioethanol from sugar (first generation bio-ethanol) hasethical problems.
Current research:Bio-ethanol from cellulosic waste (straw, wood, paper)Requires enzymes. (e.g. Simultaneous saccharification/ fermentation)
![Page 61: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/61.jpg)
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? :
1) Simple metabolism → fast degradation2) 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 strains5) Production of inhibitory acid (ph <5)
Examples: Milk, whole meal flour, vegetables,
![Page 62: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/62.jpg)
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)
![Page 63: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/63.jpg)
103 20 103
123 123
20LDH LDH
lactate
ATPATP
246
246
20
103123
= glucose
= 2 red. equiv.
= pyruvate
= lactate
Homolactic Fermentation- Electron and carbon flow -
LDH = lactate dehydrogenase
![Page 64: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/64.jpg)
O CH
C
H C H
H C H
H
Homo-lactic Fermentation- Electron and carbon flow -
O.S.: 0 → 4 electrons
O.S.: -3 → 7 electrons
O.S.: +3 → 1 electron
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
![Page 65: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/65.jpg)
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•Don’t degrade proteins•Don’t 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). •
![Page 66: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/66.jpg)
205
20
103
123
20
ATP
246 246
01
103
122
= glucose
= CO2.
= pyruvate
=acetate
= ethanol
122
205= ribose
123= lactate
8220
01 20= 2 red. equiv.
82
Heterolactic FermentationPhosphoketolase pathway
Phosphoketolase pathway = combination of Pentosephosphate cycle and FBP pathway
![Page 67: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/67.jpg)
205
20
103
123
20
ATP
246 246
01
103
122
= glucose
= CO2.
= pyruvate
=acetate
= ethanol
122
205= ribose
123= lactate
8220
01 20= 2 red. equiv.
82
Heterolactic FermentationPhosphoketolase pathway
Presence of oxygen → lactate, acetate and CO2 production → 1 additional ATP from acetokinase. No ETP
![Page 68: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/68.jpg)
Heterolactic FermentationOrganisms: 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).
![Page 69: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/69.jpg)
Application of Lactic FermentationSilage: Lactic acid fermentation of fodder materialProcess:
1) partial drying of fodder2) shredding3) Rapid filling of silo (1 or 2 days)4) packing as densely as possible5) Compressing6) Sealing airtight7) Additives (germination inhibitors, sugars, organic acids)8) Avoid contamination with decaying fodder (Clostridia,
proteolytic bacteria)
Nutrient loss:1. drying of fodder hay (25%), 2. ensilaging (10%) (2ATP out of 38)
![Page 70: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/70.jpg)
Applications of Lactic FermentationSauerkraut
In principle identical to silage with following modifications:
1) White cabbage as the only plant material2) Cabbage mixed with NaCl (2 – 2.5%)3) Capacity of vessels (concrete, wood) up to 100 tons4) Incubation (18oC to 20oC) for 4 weeks5) Recirculation of brine by pumping for process monitoring (acids)6) About 1.5% lactic acid produced7) 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
![Page 71: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/71.jpg)
Applications of Lactic FermentationSauerkraut
Similar to silage with following modifications:1) White cabbage as the only plant material2) Cabbage mixed with NaCl (2 – 2.5%)3) Capacity of vessels (concrete, wood) up to
100 tons4) Incubation (18oC to 20oC) for 4 weeks5) Recirculation of brine by pumping for
process monitoring (acids)6) About 1.5% lactic acid produced7) 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
Brin
e R
ecyc
le
![Page 72: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/72.jpg)
Brin
e R
ecyc
le
Applications of Lactic Fermentation
![Page 73: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/73.jpg)
Applications of Lactic FermentationOlives
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 >0.5%
7) Wooden barrels or plastic tanks
![Page 74: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/74.jpg)
Pickled Gherkins
1. Cover gherkins in 3% salt brine (NaCl)2. Add spices, herbs, dill3. Irradiate surface (UV) and close vessel4. After 3 – 6 weeks 3% lactic acid is produced5. Fermentation pattern like silage
![Page 75: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/75.jpg)
Applications of Lactic FermentationTechnical 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
![Page 76: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/76.jpg)
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
![Page 77: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/77.jpg)
Cheese Production Milk
HomogenisePasteurise
Add Rennet*Add starter culture(S. cremoris, S. lactis,L. bulgaricus,S. thermophilusYougurt (430°)
Curdling**StirringSettling
Heat treatment(600°)Kneading Whey
Scolding***CoolingWashingSalting
WheyQuarkFromage frais(acidic paste)
Cottage cheese(granular)
PressuringMaturing
BrieEdamer
Cheddar* Proteolytic enzyme** Coagulating*** Heated stirring
![Page 78: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/78.jpg)
20
123
20ATP
143
123 123
143
82
01
LDH
PDH
Propanoate Formation From Lactate1. Acryloyl pathway (Clostridium propionicum)
The 4 reducing equivalents from lactate oxidation to acetateare merely “dumped” onto two further moles of lactate(dismutation, disproportionation)
Enzymes: Lactate DH, Pyruvate DH, Propionate DH (PrDH)
PrDH
![Page 79: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/79.jpg)
20
123
20ATP
143
123 123
143
82
01
LDH
PDH
Propanoate Formation From Lactate1. Acryloyl pathway (Clostridium propionicum)
PrDH
Energetic benefit?
The excretion of acetate gains 1 ATP (acetate kniase),
Thus 1/3 ATP/lactate metabolised.
How to generate ATP from acetate excretionPhosphate Acetyl transferase:Acetate~CoA + Pi → Acetyl-P + CoAAcetokinase:Acetyl-P + ADP → Acetate + ATP
![Page 80: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/80.jpg)
Propanoate Formation From Lactate
2. Methyl-Malonyl-Pathway (Propionibacteria)
• 2 reducing equivalents from lactate oxidation (exactly: PDHand ferredoxin as e- carrier) are transferred via electrontransport 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 respirationHomoacetogenesis is another example
![Page 81: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/81.jpg)
20
144
20ATP
143
123 123
143
82
01
LDH
PDH
124
104
103
123
ATP
FdETC
Vit B12
01
20
123
103
104
= lactate
= pyruvate
= OAA
143= propionate
124 = fumarate (malate)
144= succinate
Propanoate Formation From Lactate2. Methyl-Malonyl-Pathway (Propionibacteria)
![Page 82: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/82.jpg)
Propionic Fermentation of Glucose
![Page 83: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/83.jpg)
Propionic Fermentation of Glucose
![Page 84: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/84.jpg)
Propionic Fermentation of Glucose
![Page 85: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/85.jpg)
Butyric Fermentation
![Page 86: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/86.jpg)
Acetone Butanol fermentation
![Page 87: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/87.jpg)
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.
![Page 88: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/88.jpg)
Homoacetogenesis
![Page 89: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/89.jpg)
• Specific growth rate u in chemostat culture• Get the D from F/V• D=u
![Page 90: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/90.jpg)
• E- acceptor from NADH in fermentations• For example acetaldehyde in ethanolic
ferm
![Page 91: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/91.jpg)
• 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
![Page 92: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/92.jpg)
• 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
![Page 93: 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](https://reader036.vdocuments.us/reader036/viewer/2022062523/5a4d1b0e7f8b9ab05998d8ba/html5/thumbnails/93.jpg)
• 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