bioprocess reaction
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Chapter 2: BioprocessReaction
CBB 20104
CONTENT MAJOR METABOLIC PATHWAYS
INTRODUCTION TO METABOLISM
GLUCOSE METABOLISM
GLYCOLYSIS, KREBS CYCLE, RESPIRATION
BIOSYSTHESIS
FERMENTATION
BIOTRANSFORMATION
BIOCONVERSION OPERATING CONSIDERATIONS FOR BIOREACTORS
FOR SUSPENSION AND IMMOBILIZED CULTURES
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MAJOR METABOLIC PATHWAYS
INTRODUCTION
Metabolismis the collection of enzyme -catalyzedreactions that convert substrates that are externalto the cell into various internal products
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CHARACTERISTICS OF METABOLISM
Varies from organisms to organism
Many common characteristics
Affected by environmental conditions
O2 availability: Saccharomyces cerevisiae
Aerobic growth on glucosemore yeast cells
Anaerobic growth on glucose ethanol Control of metabolism is important in bioprocesses
Catabolism
Metabolic reactions in the cell that degrade a substrateinto smaller / simpler products.
Glucose CO2 + H20
Produces energy for the cell
Anabolism
Metabolic reactions that result in the synthesis oflarger /more complex molecules
Glucose to glycogen
Requires energy
TYPES OF METABOLISM
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BIOENERGETICSThe source of energy to fuel cellular metabolsim is
reduced forms of carbon (sugars, hydrocarbons, etc.)
The Sun is the ultimate source via the process ofPhotosynthesis in plants
CO2 + H2O + hv CH2O + O2
Figure 5.1: Classes of Reactions
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ATP -Adenosine Triphosphate
Catabolism of carbon-containing substrates
generates high energy biomolecules
ATP -Reactions
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ATP: Energy Currency of the Cell (Fig.5.2)
NAD+ and NADP + (Fig. 5.3)
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GLUCOSE METABOLISM:Cata bolic Pa thways of Prim a ry Im portan ce
1. Glycolysis:from glucose to pyruvate.
2. Krebs or tricarboxylicacid (TCA) cycle forconversion of pyruvateto CO2.
3. Respiration orelectron transpor t chain forformation of ATP by transferring electrons fromNADH to an electron acceptor (O2under aerobicconditions).
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Glycolysis: in Eucaryotes
Krebs or TCA Cycle In Mitochondria of eucaryotes
provides e- (NADH) and ultimately energy (ATP) forbiosynthesis
provides intermediates for amino acid synthesis
generates energy (GTP)
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Krebs or TCA Cycle
Krebs or TCA Cycle
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Complete Oxidation of Glucose
Energetics of Glucose Oxidation
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ATP Yields
Respiration
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Respiration
Goals of Respiration
1. Regenerate NAD+
2. Generate ATP
Oxidative
Phosphorylation
BIOSYNTHESIS The EMP pathway and TCA cycle are critical
catabolic pathways and also provide important
precursors for the biosnythesis of amino acids,
nucleic acids, lipids and polysaccharides.
The Hexose - Monophosphate pathway (HMP)
is used for biosynthesis
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HMPPathway
*
*
*
Amino Acids by Various Pathways
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Fermentation:No TCA Cycle or Respiration
Products fromfermentation
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Metabolic Engineering (ME)
Repeated mutations were necessary to create strains of the mold
Penicillium chrysogenum which produce high titers of penicillin; that becamethe foundation of a commercial process and changed human health
care. Radiation and chemical agents were employed
by investigators to induce mutations in the
microorganism.
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Scope of m e tab olic e ngine ering
Modify host cells, host multicellular organisms, orproduct
Improved production, in selectivity or in quantity, ofchemicals already produced by the host organism
Extended substrate range for growth and productformation
Addition of new catabolic activities for degradation oftoxic chemicals
Production of chemicals new to the host organism Modification of cell properties
Ge nera l m eth odology of m e ta bolicengineering
1. Identify the target phenotype or trait
2. Increase the frequency of occurrence of gene(s) that may confer thephenotype
Increase the mutation frequency in producing cells by Mutagentreatment (UV, X-ray, chemical mutagen) (Classical method)
Introduce additional gene(s) (that may already exist or absent in the hostcell) known to give cells the desired properties (Genetic Engineering)
Introduce genetic element to inactivate or activate the gene by randominsertion of extra sequence
3. Identify the mutants (clones) that have the desired trait.
Two general means
Screening
Selection
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Stra tegies of im proving produ ction Typical objectives of metabolic engineering for chemical
production formation
Modify the pathway
Amplify the rate limiting enzyme
Redirect the flux at the divergent branch (or node) of the pathway
Remodel the regulatory element of the protein by protein engineeringor using a heterologous enzyme
Replace an enzyme(s) that is energetically or kinetically more
favorable. Amplify the first enzyme in the pathways, then identify the potential
rate limiting steps
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Enhancing the precursor and energetic supply byengineering central metabolism
Engineering the transport system
Engineering the substrate and precursor uptake
Increase the rate
Change the specificity to use new substrate of new precursor
Engineering the product secretion
Engineering the tolerance to its own product or highsubstrate concentration
Decouple the growth and production
Need biomass for product formation
Too much biomass diverts the sources if the objective is toproduce the product.
Stra tegies of im proving production
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BIOPROCESS REACTIONS
BIOPROCESS REACTION
Fermentation
Biotransformation
Bioconversion
Bioremediation
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FERMENTATION
Definition:microbial process in which enzymaticallycontrolled transformations of organic compounds occur
Fermentation has been practiced for years and hasresulted in foods such as bread, wine, and beer
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Some important fermentation productsProduct 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
-amylase Bacillus subtilis Starch hydrolysis
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The Range Of Fermentation Processes
There are four major groups of commercially importantfermentations :
Those that produce microbial cells (or biomass) as theproduct (Bakers yeast and food)
Those that produce microbial enzymes(Amylase)
Those that produce microbial metabolites (Ethanol,Citric acid)
Those that modify a compound which is added to the
fermentation the transformation processes (ethanol toacetic acid)
The fermentation plant
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The fermentation plant cont..
The component parts of a fermentation process
The formulation of medium
Sterilizing the medium
Seed fermenter
Production fermenter
Extraction and purification
Disposal effluent
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Media for Industrial FermentationsThe media is the feed solution
It must contain the essential nutrients needed for the microbeto grow
Factors of consideration when choosing mediaQuality consistence and availability
Ensure there are no problems with Media Prep or other aspectsof production process
Ex. Cane molasses, beet molasses, cereal grains
SterilizationSterilizing the feed solution is essential because the media
cannot contain foreign microbes because this could severelyhinder the growth of the production microbe
Most popular method is heat sterilization of the feed solution
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The Development of Inocula forIndustrial FermentationsThe inoculumis the starter culture that is injected into
the fermenter It must be of sufficient size for optimal growth kinetics
Since the production fermenter in industrialfermentations is so large, the inoculum volume has to bequite large
A seed fermenter is usually required to produce theinoculum volume
The seed fermenters purpose is not to produce product butto prepare inoculum
Schematic diagram
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BIOTRANSFORMATION
Definition-biological process whereby an organiccompound is modified into a recoverable product bysimple, chemically defined reactions catalyzed by enzymescontained in the cells.
Differ from fermentation results from complexbiosynthetic machinery primary and secondary metabolites
Mechanism-substrate added to microbes to transform.
Examples: production of steroids, conversion of antibiotics
and prostaglandins.
The essential difference between fermentation and
biotransformation is that there are several catalytic
steps between the substrate and the product in
fermentation while there is only one or two in a
biotransformation.
The distinction is also in the fact that the chemical
structures of the substrate and the product resemble
one another in a biotransformation, but notnecessarily in fermentation
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Example of biotransformation
D- glucoseChemical
D- sorbitol
Acetobacter
L- sorbitol
Ascorbic acid
Chemical
The world of biotransformation
Chemical modification (or modifications) made by an organism ona chemical compound.
If this modification ends in mineral compounds like CO2, NH3+
or H2O, the biotransformation is called mineralisation.
Biotransformation means chemical alteration of chemicalssuch as(but not limited to) nutrients, amino acids, toxins, or drugs in thebody. It is also needed to render nonpolar compounds polar sothat they are not reabsorbed in renal tubules and are excreted.
Biological process obey law of chemistry
Important when high specificity required Process condition: low temperature, low pressure and aqueous
Dilution is main disadvantage
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BIOCONVERSION
Using microorganism to biocatalyze specific chemical reactionbeyond the capabilities of organic chemistry
Involves growth in fermentors with specific condition
After process, desired product extract and purified.
Glucose to fructose by glucose isomerase
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Diagram
BIOREMEDIATION
A process that uses naturally occurring or geneticallyengineered microorganisms such as yeast, fungi andbacteria to transform harmful substances into less toxicor nontoxic compounds
Degrade contaminants as a source of carbon and energysources
Application-decomposting waste landfills
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Advantages and disadvantages
Advantages: Bioremediation is ecologically sound, naturalprocess; it destroys target chemicals at the contaminationsite, less expensive
Disadvantages: using bioremediation often takes longer thanother remedial method such as excavation or incineration
OPERATING CONSIDERATIONS FORBIOREACTORS FOR SUSPENSIONAND IMMOBILIZED CULTURES
Cultivation method
Batch and continuous reactors
Immobilized cell systems
Solid state fermentations
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CHOOSING THE CULTIVATION METHOD
BATCHCONTINUOUS
Most of commercial bioprocesses are batch systems
Why ?
Productivity
Many secondary products are not made by growing cells;growth represses product formation.
Under such circumstances, product is made only at low dilutionrates
For secondary products, the productivity in a batch reactor maysignificantly exceed that in a simple chemostat
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Genetic instability
Biocatalyst has undergone extensive selection
These highly bred organisms often grow less well than the parentalstrain
Back mutation from the productivespecialized strain to one similarto the less productive parental strain is always present for chemostat
In the chemostat, the less productive variant will become dominant,decreasing productivity
Operability and reliability
Batchcultures can suffer great variability from one run toanother
Variations in product quality and concentration create problemsin downstream processing and are undesirable
However, long term continuous culture can be problematic;pumps may break, controllers may fail and so on
Maintenance of sterility can be very difficult to achieve for
periods of month and the consequences of a loss of sterility aremore severe than with batcch culture
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Market economics
Many fermentations product are required in small amounts, anddemand is difficult to project
Batch process provide much greater flexibility
The same reactor can be used for two months to make productA and the next three for product B and the rest of the year forproduct C
Most bioprocesses are based on batch reactors
Continuous systems are used to make single cell protein(SCP)
Modified forms of continuous culture are used in waste
treatment
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BATCH AND CONTINUOUSREACTORS
Batch: Media and cells are added to the reactor and it is run untila predetermined set point (i.e. time, concentration). Thebioreactor has a constant volume (the initial volume).
Fed-Batch: The bioreactor is a batch process in the beginning and aftera certain point a feed input is introduced and the volume of the vesselincreases.
Continuous: The bioreactor starts with an initial volume and media isconstantly introduced and product is constantly taken out. The inputsand outputs are at the same rate, so the volume always remains the same.
Modifying batch and continuous
reactors Chemostat with recycle
Multistage chemostat systems
Fed batch operation
P10P2
F, X0 V,
X1
F, X2
F+FR,
X1
FR, XR
Growth stage Product formation stage
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IMMOBILIZED CELL SYSTEM
Restriction of cell mobility within a confined space Potential Advantages:
Provides high cell concentrations per unit of reactorvolume.
Eliminates the need for costly cell recovery andrecycle.
May allow very high volumetric productivities.
May provide higher product yields, genetic stability,
and shear damage protection.May provide favorable microenvironments such as
cell-cell contact, nutrient-product gradients, and pHgradients resulting in higher yields.
IMMOBILIZED CELL SYSTEM
Potential Disadvantages/Problems:
If cells are growing (as opposed to being in stationaryphase) and/or evolve gas (CO2), physical disruption ofimmobilization matrix could result.
Products must be excreted from the cell to berecovered easily.
Mass transfer limitations may occur as in immobilizedenzyme systems.
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METHODS OF IMMOBILIZATION
Active Immobilization:
1. Entrapment in a Porous Matrix:
METHODS OF IMMOBILIZATION
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METHODS OF IMMOBILIZATION
METHODS OF IMMOBILIZATION
Active Immobilization:
2. Cell Binding to Inert Supports::
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METHODS OF IMMOBILIZATION
METHODS OF IMMOBILIZATION
Binding Forces:
Covalent Bonding: (review enzyme covalent bonding)
Support materials: CMC-carbodiimide
support functional groups
-OH, -NH2, -COOH
Binding to proteins on cell surface
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OVERVIEW OF ACTIVE CELLIMMOBILIZATION METHODS
PASSIVE IMMOBILIZATION: BIOFILMS
The term biofilm refers to the multilayer growth of cellson solid support surfaces
Biofilms are micro-colonies of microbial cells attached toa surface and encased in adhesive polysaccharidessecreted by the cells
Biofilms trap nutrients for cell growth, and help prevent
detachment of cells on surfaces in flowing systems
Basic biofilm formation process involves attachment,colonization and development
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PASSIVE IMMOBILIZATION (BIOFILMS)
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Biofilm formation is common in industrial fermentationsystems, such as biological wastewater treatment andmold fermentations
In mixed culture microbial films, the presence of somepolymer-producing organisms facilitates biofilmformation and enhances the stability of the biofilms
Micro-environmental conditions inside a thick biofilm
vary with position, and affect the physiology of the cells
IMMOBILIZED CELL BIOREACTORS
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SOLID STATE FERMENTATION
Solid state fermentations (SSFs) involve solid substratesat low moisture levels or water activities
The water content of a typical submergedfermentation is >95%
The water content of a typical solid state fermentationis typically between 40-80%
usually used for the fermentation of agriculturalproducts or foods, such as rice, wheat, barley, corn and
soybeansThe low moisture levels of SSFs acts as a powerful
selection pressure for the growth of mycelial organisms
Advantages of SSFs overconventional submerged fermentations
The small volume of fermentation mash or rectorvolume results in lower capital and operating costs
A lower chance of contamination due to low moisturelevels
Ease of product separation
Energy efficiency
Allows the development of fully differentiatedstructures, which is critical in some cases to productformation
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Disadvantage is the heterogeneous nature of the
media, due to poor mixing characteristics
Results in control problems (pH, DO, temperature)within the fermentation mash
For large fermentation mash volumes, it can be difficult toprovide sufficient mixing to prevent concentrationgradients from forming
At high agitation speeds, mycelial cells may be damaged
Rotary-tray or rotating-drum fermenters are often used
to provide gentle yet adequate agitation in SSFs
Some Traditional FoodFermentations
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END OF CHAPTER 2
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