1. The document discusses the topics of fermentation technology and processes. It covers definitions of fermentation, important fermentation products, fermenter types, medium composition, inoculation, and microbial rates and stoichiometry.
2. Key aspects of fermentation covered include the use of stirred tank bioreactors, types of fermenters including batch, fed-batch and continuous reactors, and important industrial fermentation products like ethanol, lactic acid, and antibiotics.
3. Microbial rates are quantified as specific rates and yields, and stoichiometric equations are used to represent microbial growth coupling catabolism and anabolism based on substrate utilization and energy generation.
Upon the evolution brought about in the fermentation technology resulted out into various methodologies for optimization of the product yield by economical consumption of the substrates. Eventually, these ventures led for the development of technologies classified into as Submerged and Solid State technologies and the latter one being the concept of interest whose detailed view will be provided in the following presentation
Upon the evolution brought about in the fermentation technology resulted out into various methodologies for optimization of the product yield by economical consumption of the substrates. Eventually, these ventures led for the development of technologies classified into as Submerged and Solid State technologies and the latter one being the concept of interest whose detailed view will be provided in the following presentation
The fermentation industry is composed of five major bio-ingredient categories.
They are:
- Proteins & amino acids.
- Organic acids.
- Antibiotics.
- Enzymes.
- Vitamins & hormones.
Optimum balance of the media is mandatory for cells propagation and for the maximum production of target metabolite (end-product).
Fermentation media
Media compositions:
- Carbon source.
- Nitrogen source.
- Minerals.
- Growth factors.
- Precursors (mutants).
Types of fermentation
Solid State fermentation (SSF).
Liquid State fermentation (LSF) Surface culture & submerged culture
Production of secondary metabolites : enzymes which involves the upstream technological process
Introduction
History
Process involved
Contribution of different micro-organisms
Flowchart
Example: Methods Production of Amyalse in industrial view
Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste.
Immobilization of enzymes refers to the technique of confining/anchoring the enzymes in or on an inert support for their stability & functional reuse.
this slide is about the two most vastly used reactors i.e., batch and continuous.
A PERFECT BLEND OF INDUSTRIAL AND LABORATORY INFORMATION WITH FIRST HAND TECHNIQUES EXPLAINED IN DETAIL ABOUT VARIOUS FILTRATION TECHNIQUES, CHROMATOGRAPHY TECHNIQUES AND SEPRATION AND CELL LYSIS TECHNIQUE WITH ALL THE BASIC INFORMATION TO BEGINNERS
The fermentation industry is composed of five major bio-ingredient categories.
They are:
- Proteins & amino acids.
- Organic acids.
- Antibiotics.
- Enzymes.
- Vitamins & hormones.
Optimum balance of the media is mandatory for cells propagation and for the maximum production of target metabolite (end-product).
Fermentation media
Media compositions:
- Carbon source.
- Nitrogen source.
- Minerals.
- Growth factors.
- Precursors (mutants).
Types of fermentation
Solid State fermentation (SSF).
Liquid State fermentation (LSF) Surface culture & submerged culture
Production of secondary metabolites : enzymes which involves the upstream technological process
Introduction
History
Process involved
Contribution of different micro-organisms
Flowchart
Example: Methods Production of Amyalse in industrial view
Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste.
Immobilization of enzymes refers to the technique of confining/anchoring the enzymes in or on an inert support for their stability & functional reuse.
this slide is about the two most vastly used reactors i.e., batch and continuous.
A PERFECT BLEND OF INDUSTRIAL AND LABORATORY INFORMATION WITH FIRST HAND TECHNIQUES EXPLAINED IN DETAIL ABOUT VARIOUS FILTRATION TECHNIQUES, CHROMATOGRAPHY TECHNIQUES AND SEPRATION AND CELL LYSIS TECHNIQUE WITH ALL THE BASIC INFORMATION TO BEGINNERS
CH-3. Anaerobic treatment of wastewaterTadviDevarshi
Anaerobic treatment process, Effects of pH, temperature and other parameters on anaerobic treatment, Concept of anaerobic contact process, anaerobic filter, anaerobic fixed film reactor, fluidized bed and expanded bed reactors and up flow anaerobic sludge blanket (UASB) reactor.
Master Thesis Final Project Presentation.
Title: Microalgae growth and biomass-to-synthetic natural gas conversion through hydrothermal gasification: dynamic modeling of cultivation phase in open pond and closed reactor
Microbial catalysis of syngas fermentation into biofuels precursors - An expe...Pratap Jung Rai
Search for environment-friendly sustainable energy sources is of global interest due to continuous depletion of fossil fuels resources and excessive carbon dioxide emissions. Syngas fermentation is one of the promising sustainable alternative for liquid biofuel and chemical production from energy content wastes/byproducts. This study mainly focuses on acetic acid and ethanol production via fermentation, using hydrogen and carbon dioxide as substrates to mimic syngas. A laboratory scale, batch fermentation was performed at different headspace pressure ranged from 0.29 to 1.51 bar, 1200 rpm stirrer speed, and 22±1.4ºC.
Formation of acetic acid and ethanol were found significant. The maximum acetic acid concentration 68 mmol/L was obtained at 1176 hours and 1.12 bar headspace pressure. However, maximum ethanol concentration of 15 pA*s was found at 1297 hours and 1.51 bar headspace pressure. Ethanol consumption was observed during first 553 hours. Maximum H2 consumption rate was 0.153 mmol/h•gVS during 478-527 hours at 1.12 bar headspace pressure, which was 51 times higher than that obtained during first 71 hours at 0.29 bar headspace pressure (0.003 mmol/h• gVS). The total consumed hydrogen gas measure as COD (CODHydrogen) was equivalent to the increase in bulk liquid COD, 11.02 gCOD and 11.44 gCOD; in which 68% of CODHydrogen was converted to acetic acid (7.44 gCOD). A significant influence of headspace pressure and dissolved hydrogen concentration were observed on the volumetric mass (H2) transfer coefficient (kLa) and the solubility of hydrogen in the inoculum (CH). The maximum kLa and CH of 0.082 h-1 (R2 = 0.995) and 1.2 10-3 mol/L were found at 1.12 bar headspace pressure and 89 mmol/L dissolved hydrogen concentration, respectively. The calculated biomass yields ranged from 0.001-0.066 and 0.001-0.059 gVSS/gCOD, for acetic acid and ethanol formation, respectively, when the assumption of free energy efficiency use in growth was changed from 0.1 to 1.
Acetic acid and ethanol were dominant final product whereas other organic acids were almost constant and insignificant throughout the experiment. This implies that the microbial fermentation of hydrogen and carbon dioxide at headspace pressure ranged from 0.29-1.51 bar, 1200 rpm stirrer speed, and 22±1.4ºC, can be performed with digested food waste sludge for efficient acetic acid and ethanol production.
2. 2
Course content
I. Introduction
II. General aspects of fermentation processes
III. Quantification of microbial rates
IV. Stoichiometry of microbial growth and product
formation
V. Black box growth
VI. Growth and product formation
VII. Heat transfer in fermentation
VIII. Mass transfer in fermentation
IX. Unit operations in fermentation (introduction to
downstream processing)
X. Bioreactor
4. 4
What is fermentation?
• Pasteur’s definition: “life without air”, anaerobe
red ox reactions in organisms
• New definition: a form of metabolism in which the
end products could be further oxidized
For example: a yeast cell obtains 2 molecules of
ATP per molecule of glucose when it ferments it
to ethanol
5. 5
What is fermentation techniques (1)?
Techniques for large-scale production of microbial products.
It must both provide an optimum environment for the
microbial synthesis of the desired product and be
economically feasible on a large scale. They can be divided
into surface (emersion) and submersion techniques. The latter
may be run in batch, fed batch, continuous reactors
In the surface techniques, the microorganisms are cultivated
on the surface of a liquid or solid substrate. These techniques
are very complicated and rarely used in industry
6. 6
What is fermentation techniques (2)?
In the submersion processes, the microorganisms grow in a
liquid medium. Except in traditional beer and wine
fermentation, the medium is held in fermenters and stirred to
obtain a homogeneous distribution of cells and medium. Most
processes are aerobic, and for these the medium must be
vigorously aerated. All important industrial processes
(production of biomass and protein, antibiotics, enzymes and
sewage treatment) are carried out by submersion processes.
7. 7
Some important fermentation products
Product Organism Use
Ethanol Saccharomyces
cerevisiae
Industrial solvents,
beverages
Glycerol Saccharomyces
cerevisiae
Production of
explosives
Lactic acid Lactobacillus
bulgaricus
Food and
pharmaceutical
Acetone and
butanol
Clostridium
acetobutylicum
Solvents
α-amylase Bacillus subtilis Starch hydrolysis
13. 13
Fermenter
The heart of the fermentation process is the fermenter.
In general:
• Stirred vessel, H/D ≈ 3
• Volume 1-1000 m3
(80 % filled)
• Biomass up to 100 kg dry weight/m3
•Product 10 mg/l –200 g/l
14. 14
Types of fermenter
• Simple fermenters (batch and continuous)
• Fed batch fermenter
• Air-lift or bubble fermenter
• Cyclone column fermenter
• Tower fermenter
• Other more advanced systems, etc
The size is few liters (laboratory use) - >500 m3
(industrial applications)
15. 15
Cross section of a fermenter for Penicillin production ( Copyright:
http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)
16. 16
Cross section of a fermenter for Penicillin production ( Copyright:
http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)
17. 17
Flow sheet of a multipurpose fermenter and its
auxiliary equipment
18. 18
Fermentation medium
• Define medium nutritional, hormonal, and
substratum requirement of cells
• In most cases, the medium is independent of the
bioreactor design and process parameters
• The type: complex and synthetic medium (mineral
medium)
• Even small modifications in the medium could
change cell line stability, product quality, yield,
operational parameters, and downstream processing.
19. 19
Medium composition
Fermentation medium consists of:
• Macronutrients (C, H, N, S, P, Mg sources water,
sugars, lipid, amino acids, salt minerals)
• Micronutrients (trace elements/ metals, vitamins)
• Additional factors: growth factors, attachment
proteins, transport proteins, etc)
For aerobic culture, oxygen is sparged
20. 20
Inoculums
Incoculum is the substance/ cell culture that is
introduced to the medium. The cell then grow in the
medium, conducting metabolisms.
Inoculum is prepared for the inoculation before the
fermentation starts.
It needs to be optimized for better performance:
• Adaptation in the medium
• Mutation (DNA recombinant, radiation, chemical
addition)
23. 23
Microbial rates of consumption or production
C, N, P, S source
H2O
H+
O2
heat
product
CO2
biomass
24. 24
What are the value of rates?
Rates of consumption or production are obtained from
mass balance over reactors
Mass balance over reactors
Transport + conversion = accumulation
(in – out) + (production – consumption) = accumulation
Batch: transport in = transport out = 0
Chemostat: accumulation = 0, steady state
Fed batch: transport out = 0
25. 25
How are rates defined?
Rate (ri) = amount i per hour / volume of reactor
Biomass specific rate (qi)
qi =amount per hour / amount of organism in reactor
Thus:
Substrate (-rS) = (-qS)CX
Biomass rX = µCX
Product rP = qPCX
reactorm
hourikg
−3
/.
Xkg
hourikg
.
/.
ri = qi CX
26. 26
Yield = ratio of rates
Yij = i
j
Xi
Xj
i
j
q
q
Cq
Cq
r
r
irate
jrate
===
.
.
YSX = rate of biomass production / rate of substrate
consumption [g biomass/g substrate]
YOX = rate of biomass production / rate of oxygen
consumption [g biomass/g oxygen]
28. 28
Introduction
Cell growth and product formation are complex processes
reflecting the overall kinetics and stoichiometry of the
thousands of intracellular reactions that can be observed within
a cell.
Thermodynamic limit is important for process optimization.
The complexity of the reactions can be represented by a simple
pseudochemical equation.
Several definitions have to be well understood before studying
this chapter, for example: YSX
max
, YATP X, YOX, maintenance
coefficient based on substrate (ms).
29. 29
Composition of biomass
Molecules
• Protein 30-60 %
• Carbohydrate 5-30 %
• Lipid 5-10 %
• DNA 1 %
• RNA 5-15 %
• Ash (P, K+
, Mg2+
, etc)
• Elements
• C 40-50 %
• H 7-10 %
• O 20-30 %
• N 5-10 %
• P 1-3 %
• Ash 3-10%
Typical composition biomass formula: C1H1.8O0.5N0.2
Suppose 1 kg dry biomass contains 5 % ash, what is the
amount of organic matter in C-mol biomass?
30. 30
Anabolism
Amino acids protein
Sugars carbohydrate
Fatty acids lipids
Nucleotides DNA, RNA
Sum of all reactions gives the anabolic reaction
(…)C-source + (…)N-source + (…) P-source + O-source
C1H1.8O0.5N0.2 + (…)H2O + (…)CO2
Thermodynamically, energy is needed. Also for cells
maintenance
energy
31. 31
Catabolism
Catabolism generates the energy needed for anabolism and
maintenance. It consist of electron donor couple and
electron donor acceptor couple
For example:
• Glucose + (…)O2 (…)HCO3
-
+ H2O
donor couple: glucose/HCO3
-
acceptor couple: O2/H2O
• Glucose (…)HCO3
-
+ (…)ethanol
donor couple: glucose/HCO3
-
acceptor couple: CO2/ethanol
The catabolism produces Gibbs energy (∆Gcat.reaction)
32. 32
Coupled anabolism/catabolism
C-source (anabolism) and electron-donor (catabolism) are
often the same (e.g. organic substrate)
Only a fraction of the substrate ends in biomass as C-source,
while the rest is catabolized as electron-donor to provide
energy for anabolism and maintenance
YSX is the result of anabolic/catabolic coupling.
33. 33
Several examples stoichiometry of growth
Aerobic growth on oxalate
5.815 C2O4
2-
+ 0.2 NH4
+
+ 1.8575 O2 + 0.8 H+
+ 5.415 H2O
C1H1.8O0.5N0.2 + 10.63 HCO3
-
What is C-source? N-source? Electron donor? Electron
acceptor?
YSX = 1 C-mol X / 5.815 mol oxalate = 1 C-mol X / 11.63 C-
mol oxalate
Catabolic reaction for oxalate:
C2O4
2-
+ 0.5 O2 + H2O 2HCO3
-
or H2C2O4 + 0.5 O2 H2O + 2CO2
35. 35
Microbial growth stoichiometry using
conservation principles
The general equation for growth stoichiometry
-1/YSX substrate + (…)N-source + (…)electron acceptor +
(…)H2O + (…)HCO3
-
+ (…)H+
+ C1H1.8O0.5N0.2 +
(…)oxidized substrate + (…)reduced acceptor
(…) > 0 for product, (…) < 0 for reactant
Note:
1. N-source, H2O, HCO3
-
, H+
and biomass are always present
2. Only substrate and electron acceptor are case specific
3. YSX is mostly available, all other coefficients follow the
element or charge conservation
36. 36
Aerobic growth of Pseudomonas oxalaticus
using NH4
+
and oxalate (C2O4
2-
)
Electron donor couple?
Electron acceptor couple?
C-source? N-source?
YSX is 0.0506 gram biomass/ gram oxalate and biomass has 5 %
ash. Biomass molecular weight = 24.6 g/C-mol X
YSX = C-mol X/mol oxalate172.0
6.24
95.0*88*0506.0
=
37. 37
• Set up the general stoichiometric equation
f C2O4
2-
+ a NH4
+
+ b H+
+ c O2 + d H2O C1H1.8O0.5N0.2 + e
HCO3
-
• Use YSX to calculate f
f = mol oxalate/C-mol X
• There are 5 unknowns (a, b, c, d, e) and 5 conservation
balance (C, H, O, N, charge). For example:
C : 2f = 1 + e
H? O? N? charge?
• Solve for a, b, c, d, and e!
• What is the value of respiratory quotient (RQ)? Remember
815.5
172.0
11
−=−=−
SXY
2
2
O
CO
q
q
RQ =
39. 39
What is degree of reduction (γi)?
• It is about proton-electron balance in bioreactions
• Stoichiometric quantity of compound I
• Electron content of compound i relative to reference
The references (γi = 0):
HCO3
-
/CO2
H+
/OH-
NH4
+
/NH3
SO4
2-
Fe3+
N-source for growth
atom γi
C +4
H +1
O -2
N -3
S +6
Fe +3
+ charge -1
- charge +1
NH4
+
as N-source -3
N2
as N-source 0
NO3
-
as N-source +5
40. 40
γ for compounds
For example: glucose (C6H12O6)
γ glucose = 6(4) + 12(1) + 6(-2) = 24 = 4/C-glucose
Biomass? O2? Fe2+
? Citric acid? Ethanol? Lactic acid?
γ-balance
It is used to calculate stoichiometry
It follows from conservation relations (C, H, O, N, charge, etc)
by eliminating the unknown stoichiometric coefficient for
reference compounds
It relates biomass, substrate/donor, acceptor, product
(H2O, H+
, HCO3
-
, N-source are always absent)
41. 41
Example
Catabolism of glucose to ethanol in anaerobic culture
-C6H12O6 + aC2H6O +bCO2 + cH2O +dH+
γ glucose = 24, γ ethanol = 12, γ balance = -24+12a = 0, a = 2
b, c, d follow from C,O, and charge conservation
Thus: -C6H12O6 + 2 C2H6O + 2 CO2
Try to solve:
a. Catabolism of ethanol to acetate (C2H3O2
-
) using O2/H2O
b. Catabolism of H2S to S-
using NO3
-
/NO2
-
c. Anabolic reaction, glucose as C-source and electron donor
d. Complete growth reaction, aerobic growth on oxalate
(C2O4
2-
)
42. 42
Further reading
Stoichiometry calculations in undefined chemical systems for
fermentation with complex medium, biological waste
water treatment, and soluble and non-soluble compounds
Measurements of lumped quantities:
1. TOC, Carbon balance
2. Kj-N, Kjeldahl-nitrogen for all reduced nitrogen (organic
bound and NH4
+
), N-balance
3. ThOD, COD balance (similar to γ balance)