fermentation technology, bioprocess principles

Historical developmentHistorical developmentFermentation products – wine, bread etc.,

Invention of microbes responsible for these conversion – science of microbiology

Penicillin fermentation – sterility concepts, pilot plant concepts

Single cell production by ICI – air lift fermenters, continuous cultivation, process control

Stage Main Pdts Vessel

Process control

Culture method

QCPilot plant facilities

Strain S188election

Pre 1900

Alcohol

Wooden, upto 1500 barrels capacity, Copper used in lateral breweries

Use of thermometers, hydrometer and heat exchangers

BatchVirtually nil

Nil

Pure yeast cultures used at carlsberg brewery 1886

Vinegar

Barrels, shallow trays, trickling filters

Fermentation inoculated with good vinegar

1900 - 1940

Bakers’s yeast

Steel vessel for actone / butanol

pH electrode with off-line control

Batch Virtually nil

NilPur cutures used

Stage Main Pdts Vessel

Process control

Culture method

QCPilot plant facilities

Strain S188election

1940 – date

Penicillin, streptomycin and other antibiotics

Mechanically seperated vessel, operated aseptically – true fermenters

Sterilizable pH and oxygen electrodes. Use of control loops computerized

Batch, fed-batch and continuous

Very important

Becomes common

Mutation selection and programme essentials

1964 – date SCP

Pressure cycle and pressure jet vessels developed to overcome gas and heat exchanger problems

Use of computer linked control

Continuous with medium cycle

Very important

Very important

GE

1979 – date MABs

Animal cell reators

Control and sensor

Batch, fed, continous

Very important

Very important

rDNA

Biotechnology Products Biotechnology Products

Microbial biomassMicrobial biomass EnzymesEnzymes Primary and secondary Primary and secondary metabolitesmetabolites

Recombinant productsRecombinant products BiotransformationBiotransformation

Industry Application Enzyme SourceBaking & Milling

Reduction in dough viscosity

Amylase Fungal

Brewing Mashing Amylase Fungal /. Bacterial

Laundry Detergent Protease / lipase / cellulase

Fungal / Bacterial

Leather Dehairing Protease Fungal / Bacterial

Meat Tenderization

Protease Fungal

Photography

Recovery of silver from spent film

Protease Bacterial

Soft drinks

Stabilization

Glucose oxidase, catalase

Fungal

Textiles Desizing of fabrics

Amylase Bacterial

Operations in Operations in FermentationFermentation

Inoculum preparationInoculum preparation Medium preparationMedium preparation SterilizationSterilization Cultivation Cultivation

Batch, Fed Batch, Continuous etc.,Batch, Fed Batch, Continuous etc., Downstream processingDownstream processing Effluent treatmentEffluent treatment

Fermentation Industry Fermentation Industry OverviewOverview

Fermentation bayUtilityDownstream Processing

Microbiology & Quality Control

STORES

Effluent treatment

UtilitiesUtilities Steam – 4 BAR & 1.5 BARSteam – 4 BAR & 1.5 BAR Air – 1.5 BARAir – 1.5 BAR Chilled waterChilled water Cooling waterCooling water Deionised waterDeionised water SoftwaterSoftwater MCCMCC

Fermentation bayFermentation bay Seed FermenterSeed Fermenter Main fermenterMain fermenter Acid, Alkali & Antifoam tanksAcid, Alkali & Antifoam tanks Nutrient feedingNutrient feeding Harvest tankHarvest tank

Microbiology LaboratoryMicrobiology Laboratory Media preparationMedia preparation AutoclavingAutoclaving Inoculation chamberInoculation chamber Microscopic observationMicroscopic observation Quality controlQuality control Culture stock maintenanceCulture stock maintenance

Culture Stock Culture Stock MaintenanceMaintenance

Plates – 15 daysPlates – 15 days Stab/slant – 6 monthsStab/slant – 6 months Glycerol stock – 6 monthsGlycerol stock – 6 months Lyophilisation – 15 yearsLyophilisation – 15 years Sand cultures – 15 yearsSand cultures – 15 years Liquid nitrogen storage – 15 Liquid nitrogen storage – 15 yearsyears

Culture ValidationCulture Validation

BioreactorBioreactorHow a bioreactor differs from a chemical reactor ?How a bioreactor differs from a chemical reactor ?

Both are agitated tanksBoth are agitated tanks

Bioreactor should be capable of being operated Bioreactor should be capable of being operated aseptically for number of daysaseptically for number of days

Adequate aeration and agitation should be Adequate aeration and agitation should be provided to meet the metabolic requirements provided to meet the metabolic requirements which will vary from time to timewhich will vary from time to time

Material of Material of ConstructionConstruction

Lab fermenters are made up of glassLab fermenters are made up of glass Pilot and industrial scale fermenters Pilot and industrial scale fermenters are made up of Stainless steelare made up of Stainless steel

Steel having chromium more than 4% Steel having chromium more than 4% are called us stainless steel.are called us stainless steel.

The corrosion resistance of stainless The corrosion resistance of stainless steel depend on the existence of a steel depend on the existence of a thin hydrous oxide film on the thin hydrous oxide film on the surface of the metalsurface of the metal

The composition of this film varies The composition of this film varies with different steel alloys and with different steel alloys and manufacturing processmanufacturing process

This oxide film is stabilized by This oxide film is stabilized by chromiumchromium

If the film is damaged it will repair If the film is damaged it will repair itself when exposed to air or itself when exposed to air or oxidizing agent.oxidizing agent.

Minimum amount of chromium needed to Minimum amount of chromium needed to resist corrosion will depend on the resist corrosion will depend on the corroding agent in environment (acid, corroding agent in environment (acid, alkali, salt etc)alkali, salt etc)

Increasing chromium enhances the Increasing chromium enhances the corrosion resistance.corrosion resistance.

10 – 13% chromium will develop 10 – 13% chromium will develop effective oxide filmeffective oxide film

Also addition of nickel to this high Also addition of nickel to this high percent chromium steel enhances the percent chromium steel enhances the resistance to corrosion and give resistance to corrosion and give strength.strength.

Addition of molybdenum improves the Addition of molybdenum improves the resistance of stainless steel to resistance of stainless steel to solution of salts or halogens such as solution of salts or halogens such as chlorides.chlorides.

Corrosion resistance can also be Corrosion resistance can also be increased using tungstan silicone increased using tungstan silicone etc.etc.

Most commonly used stainless steel Most commonly used stainless steel for fermentation equipment is for fermentation equipment is SS316 Cr Ni Mo : 17 12 2SS316 Cr Ni Mo : 17 12 2 SS316L Cr Ni Mo : 18 14 3SS316L Cr Ni Mo : 18 14 3 SS317 Cr Ni Mo : 17 13 4SS317 Cr Ni Mo : 17 13 4 SS304 Cr Ni : 18.5 10 SS304 Cr Ni : 18.5 10

Normally the main tank is made up Normally the main tank is made up of 316 grades and jackets are made of 316 grades and jackets are made up of 304 to reduce the cost.up of 304 to reduce the cost.

Shape of the vesselShape of the vessel

Height to Diameter ratio :Height to Diameter ratio : Height of the fermenter is one of the critical Height of the fermenter is one of the critical

parameters in the fermenter design.parameters in the fermenter design. If the height of the fermenter is more the If the height of the fermenter is more the

bubble residence time is more and better bubble residence time is more and better oxygen efficiency.oxygen efficiency.

H/D ratio of the fermenter 2 to 3 is preferred.H/D ratio of the fermenter 2 to 3 is preferred.

For Design Purpose For Design Purpose the effective the effective working volume of working volume of the reactor is the reactor is considered to be considered to be 80% of Total volume 80% of Total volume of the reactor.of the reactor.

Fermenter will be Fermenter will be running 330 days running 330 days per annum per annum

80%

Constant heat flux jacket

Cooling coil Spiral

Final half coil vessel

MixingMixing One of the major task of bioreactor is mixingOne of the major task of bioreactor is mixing Mixing is a physical operation which reduces Mixing is a physical operation which reduces

non uniformities in fluid by eliminating non uniformities in fluid by eliminating gradients of concentration, temperature and gradients of concentration, temperature and other propertiesother properties

Mixing in the Bioreactor involvesMixing in the Bioreactor involves Blending the soluble medium components eg. SugarsBlending the soluble medium components eg. Sugars Dispersing air in the form of small bubblesDispersing air in the form of small bubbles Suspension of solid particles such as cellsSuspension of solid particles such as cells Promoting heat transfer and dispersion of immiscible Promoting heat transfer and dispersion of immiscible

liquids such as antifoamliquids such as antifoam To carry out physical change – crystal formation To carry out physical change – crystal formation

Radial Radial flow with flow with bafflebaffle

Axial flow Axial flow with with bafflebaffle

Types of Types of agitatoragitator

Pitched BladeTurbinePropeller

ImpellerImpeller Propeller and pitched blade – axial flow Propeller and pitched blade – axial flow Paddles and flat blade, disk flat blade – Paddles and flat blade, disk flat blade – radial flowradial flow

Propeller: Propeller: for low viscous liquids. for low viscous liquids. Its simple and portable having tampering blade Its simple and portable having tampering blade for radial flowfor radial flow

Dimensions: diameter is 15-30% of diameter of Dimensions: diameter is 15-30% of diameter of tanktank

Small ones rotate @ full motor speed 400 - 800 Small ones rotate @ full motor speed 400 - 800 rpmrpm

Propeller @ bottom to avoid settling of heavy Propeller @ bottom to avoid settling of heavy particlesparticles

ImpellerImpeller Turbines: Turbines: Pitched blade – axial type; Pitched blade – axial type; Curved blade – Curved blade – radial typeradial type

Capable of creating vigorous mixing action Capable of creating vigorous mixing action due to centrifugal and rotational notion due to centrifugal and rotational notion generated.generated.

Dimensions: Diameter is kept 1/3Dimensions: Diameter is kept 1/3rdrd and 1/6 and 1/6thth of vessel diameter.of vessel diameter.

Blade length is 1/4Blade length is 1/4thth of the impeller diameter. of the impeller diameter.With central disc it is 1/8With central disc it is 1/8thth of impeller of impeller diameter.diameter.

Curved blade – 30-60°Curved blade – 30-60°Speed: 50 – 250 rpmSpeed: 50 – 250 rpm

ImpellerImpeller Turbines: Turbines: Viscosity – 10Viscosity – 1044 cP cP Stream divided into two portions: Stream divided into two portions: one flow to bottom and return to one flow to bottom and return to shaft and other flow upward and shaft and other flow upward and reach shaft. reach shaft.

ImpellerImpeller Paddles: Paddles: Viscosity – very high viscosity Viscosity – very high viscosity ranges.ranges.

Stream divided into two portions: Stream divided into two portions: one flow to bottom and return to one flow to bottom and return to shaft and other flow upward and shaft and other flow upward and reach shaft. reach shaft.

Multiple Multiple impellersimpellers

Stagnant Stagnant zones in big zones in big

reactorsreactors

Geometrical ratios of Geometrical ratios of FermenterFermenter

H/D H/D : 2 – 3 : 2 – 3 Imp dia P : 0.3 – 0.5 DImp dia P : 0.3 – 0.5 D Baffle Baffle : 0.1 D : 0.1 D W/P W/P : 1.5 – 2.0 : 1.5 – 2.0 P/ZP/Z : 0.5-1.0 : 0.5-1.0

Circular flowCircular flow

BaffleBaffle Normally four Baffles are installed in the Normally four Baffles are installed in the

BioreactorBioreactor Purpose of Baffle is to avoid the vortex formation Purpose of Baffle is to avoid the vortex formation

and increasing the aeration efficiencyand increasing the aeration efficiency Baffles are normally 1/10Baffles are normally 1/10thth of the dia of the vessel. of the dia of the vessel. Increasing the size of the baffle increase the Increasing the size of the baffle increase the

agitation effect marginally.agitation effect marginally. Decreasing that will have substantial drop in Decreasing that will have substantial drop in

efficiency.efficiency. Baffles should be installed such that gap exist Baffles should be installed such that gap exist

between the vessel and baffle which minimizes between the vessel and baffle which minimizes dead space and avoid microbial growthdead space and avoid microbial growth

Baffles can be Baffles can be effectively used effectively used to increase the to increase the heat transfer heat transfer efficiencyefficiency

Baffles can be Baffles can be used as level used as level indicatorsindicators

Baffles can be Baffles can be used as inlet and used as inlet and exit point in big exit point in big fermentersfermenters

Power Consumption in Agitated Power Consumption in Agitated VesselVessel

The power-number has different specifications The power-number has different specifications according to the field of application. E.g., according to the field of application. E.g., for stirrers the power number is defined as:for stirrers the power number is defined as:

NNpp = P / = P / ρρ N N33 Da Da55

Stirred vesselStirred vesselIn a cylindrical vessel stirred by a central In a cylindrical vessel stirred by a central rotating paddle, turbine or propeller, the rotating paddle, turbine or propeller, the characteristic dimension is the diameter of characteristic dimension is the diameter of the agitator . The velocity is where  is the agitator . The velocity is where  is the rotational speed. Then the Reynolds the rotational speed. Then the Reynolds number isnumber is

NNReRe = = ρρ N Da N Da22 / µ / µ

Power Consumption in Agitated Power Consumption in Agitated VesselVessel

Problem:Problem:

In a lab scale bioreactor production In a lab scale bioreactor production of gluconic acid in optimized medium of gluconic acid in optimized medium is 180 g/l. The time duration of is 180 g/l. The time duration of cultivation is 36 hours and downtime cultivation is 36 hours and downtime for cleaning, sterilization, medium for cleaning, sterilization, medium preparation etc. is 12 hours. You preparation etc. is 12 hours. You have been asked to design a have been asked to design a fermentor for the production of 2000 fermentor for the production of 2000 Tonnes per annum. State your Tonnes per annum. State your assumptions and geometrical ratios assumptions and geometrical ratios for your design?for your design?

Air FilterAir Filter

Air Inlet Line inBioreactor

Air outlet Air outlet line in line in

BioreactorBioreactor

Rupture Rupture diskdisk

Drain Drain ValveValve

Sampling Sampling valvevalve

PortsPorts Small reactor -12 mm portsSmall reactor -12 mm ports Pilot scale – 19 and 25 mm Pilot scale – 19 and 25 mm portsports

Big reactors - any size Big reactors - any size depending on the probes.depending on the probes.

INSTRUMENTATIONMeasurement type

Inline measurement : The sensor is an integrated part of the fermentation equipment and measure the process variable real time continuously.

Online measurement: Continuous/discontinuous measurement but the sensor is not an integrated part of the bioreactor but connected to the system

Offline sensor: Discontinuous measurement and the sensor is not an integrated part of the system. Samples have to be connected and measured manually

When evaluating sensors to use in When evaluating sensors to use in measurement and control it is measurement and control it is important to consider the important to consider the following:following:

Response timeResponse time Gain and sensitivityGain and sensitivity AccuracyAccuracy Ease and speed of calibrationEase and speed of calibration ReliabilityReliability Material of constructionMaterial of construction SterilizationSterilization

Bioreactor variables Physical variables

Temperature, Pressure, Flow, Agitation speed, Mass, Volume, Foam

Chemical variables pH, Dissolved oxygen, Dissolved carbon dioxide, ion concentration, substrate concentration

Biological variables Cell concentration, Protein, DNA,RNA, Enzyme activities etc

Temperature Measurement Mercury in glass thermometers, thermocouples, bimetallic thermometers, thermistors, Metal resistance thermometers etc.,

Metal resistance thermometers are most commonly used.

Mercury in glass thermometers are used for precise calibration of other measurement systems

Metal resistance thermometers

Platinum resistance thermometers are commonly used in bioreactors. (Pt 100)

Electrical resistance of platinum changes with change in temperature.

Electrical resistance wire of 100 ohms is used

This is connected to one side of Wheatstone bridge and other three sides are having known resistance.

The known electrical voltage is applied across the bridge and change in the voltage occurs when the resistance changes according to the temperature

Change in resistance 0.385 to 0.392 ohms/Change in resistance 0.385 to 0.392 ohms/ooCC

Pressure MeasurementPressure Measurement Pressure is one of the critical Pressure is one of the critical measurements mainly for the measurements mainly for the safety of the bioreactor.safety of the bioreactor.

Always bioreactors are operated Always bioreactors are operated under positive pressure i.e under positive pressure i.e pressure above atmospheric pressure above atmospheric pressure to prevent contamination pressure to prevent contamination and better oxygen transfer.and better oxygen transfer.

Pressure will buidup in the Pressure will buidup in the bioreactor during sterilizationbioreactor during sterilization

DevicesDevices C tube bourdon gauge - manualC tube bourdon gauge - manual If a wire is subject to strain its If a wire is subject to strain its electrical resistance changes.electrical resistance changes.

Flow MeasurementFlow Measurement Gas flow measurement – area rotameterGas flow measurement – area rotameter Mass flow meterMass flow meter Liquid flow – weighing the mass or Liquid flow – weighing the mass or metering pumpmetering pump

RotameterRotameterAdvantagesAdvantages:: A rotameter requires no external power or fuel, it uses only the A rotameter requires no external power or fuel, it uses only the

inherent properties of the fluid, along with gravity, to measure inherent properties of the fluid, along with gravity, to measure flow rate. flow rate.

A rotameter is also a relatively simple device that can be mass A rotameter is also a relatively simple device that can be mass manufactured out of cheap materials, allowing for widespread use manufactured out of cheap materials, allowing for widespread use in places such as third-world countries. in places such as third-world countries.

DisavantagesDisavantages:: Due to its use of gravity, a rotameter must always be vertically Due to its use of gravity, a rotameter must always be vertically

oriented and right way up, with the fluid flowing upward. oriented and right way up, with the fluid flowing upward. Due to its reliance on the ability of the fluid or gas to displace Due to its reliance on the ability of the fluid or gas to displace

the float, graduations on a given rotameter will only be accurate the float, graduations on a given rotameter will only be accurate for a given substance. The main property of importance is the for a given substance. The main property of importance is the density of the fluid; however, viscosity may also be significant. density of the fluid; however, viscosity may also be significant. Floats are ideally designed to be insensitive to viscosity; Floats are ideally designed to be insensitive to viscosity; however, this is seldom verifiable from manufacturers specs. however, this is seldom verifiable from manufacturers specs. Either separate rotameters for different densities and viscosities Either separate rotameters for different densities and viscosities may be used, or multiple scales on the same rotameter can be used. may be used, or multiple scales on the same rotameter can be used.

Rotameters normally require the use of glass (or other transparent Rotameters normally require the use of glass (or other transparent material), otherwise the user cannot see the float. This limits material), otherwise the user cannot see the float. This limits their use in many industries to benign fluids, such as water. their use in many industries to benign fluids, such as water.

Rotameters are not easily adapted for reading by machine; although Rotameters are not easily adapted for reading by machine; although magnetic floats that drive a follower outside the tube are magnetic floats that drive a follower outside the tube are availableavailable

                 Temperature at the sensors varies depending upon the mass flow

THERMAL MASS FLOWMETERTHERMAL MASS FLOWMETER

SpeedSpeed Measurement by Tachometer fixed in the Measurement by Tachometer fixed in the motor – electromagnetic voltage generationmotor – electromagnetic voltage generation

WeightWeight

Measurement by load cellMeasurement by load cell

FoamFoam Capacitance probesCapacitance probes Conductivity probesConductivity probes Thermal conductivityThermal conductivity UltrasonicUltrasonic Rotating diskRotating disk

Biomass MeasurementBiomass Measurement

Dry weightDry weight OD 600OD 600 PCVPCV Cell countCell count Fluorescence probeFluorescence probe

pH ProbepH Probe

The pH probe measures pH as the The pH probe measures pH as the activity of hydrogen ions activity of hydrogen ions surrounding a thin-walled glass surrounding a thin-walled glass bulb at its tip. The probe bulb at its tip. The probe produces a small voltage (about produces a small voltage (about 0.06 volt per pH unit) that is 0.06 volt per pH unit) that is measured and displayed as pH measured and displayed as pH units by the meter units by the meter

The reactions are as follows:The reactions are as follows:At cathode: O2 + 4H+ + 4 e- → 2H2OAt cathode: O2 + 4H+ + 4 e- → 2H2OAt anode: 2H2O → O2 + 4H+ + 4 e-At anode: 2H2O → O2 + 4H+ + 4 e-

Oxygen sensors have a thin organic membrane covering a layer of electrolyte and two metal electrodes. Oxygen diffuses through the membrane at a rate proportional to its partial pressure—the greater the oxygen partial pressure, the more oxygen diffuses through the membrane. Oxygen meters measure the current as oxygen is reduced at the cathode and more oxygen diffuses through the membrane. Since the diffusion current is directly proportional to the concentration of dissolved oxygen, the calibrated meter simply converts measured current into concentration units.