bioprocess reaction

7/21/2019 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:



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Active Immobilization:

2. Cell Binding to Inert Supports::


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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

bioprocess reaction

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