COMPONENTS OF A FERMENTER

AIM: To study the construction & control system of a fermenter. Theory: A fermenter is a system consisting of a few pieces of equipments which provide controlled environmental conditions for the growth of microbes (and/or production of specific metabolites) in liquid culture while preventing entry and growth of contaminating microbes from the outside environment. Major components of a fermenter: Base components including drive motor, heater, pumps, gas control, etc. Vessel and accessories. Peripheral equipments such as reagent bottles. Instrumentation and sensors. These components combine to perform the following functions: Facilitates the growth of a wide range of organisms. Provide operations free from contamination. Maintain a specific temperature. Provide adequate mixing and aeration. Control the pH of the culture. Allow monitoring and/or control of dissolved oxygen. Allow feeding of nutrients solutions and reagents. Provide access points for inoculation and sampling. Body Construction: For a small scale (1 to 30 dm3) glass and/or stainless steel is used because it can withstand repeated steam sterilization cycles. Two basic types are used: A glass vessel with a round or flat bottom and top flanged carrying plates. A glass cylinder with stainless steel top and bottom plates. This fermenter may be sterilized in situ. (AISI graded steel are now commonly used in fermenter construction.) Peripheral parts: Reagents pumps: Pumps are normally part of the instrumentation system for pH and antifoam control.

Peristaltic pumps are used and flow rate is usually fixed with a timed shot and delay feed system of control.

Medium feed pump and reservoir bottle: Medium feed pumps are often variable speed to give the maximum possible range of feed rates. The reservoir bottles are usually larger , but are prepared in the same as normal reagents bottle. Rota meter: A variable area flow mere which indicates the rate of gas flow into a fermenter. A pressure regulator valve before the Rota meter ensures safe operation. Structural component involve in aeration and agitation: 1) Agitator (impeller) Function: Bulk fluid and gas phase mixing Air dispersion Oxygen transfer Heat transfer Suspension of solid particles Maintaining uniform environment throughout the vessel contents. Types of agitators: Disc turbine Open turbines of variable pitch Propellers 2) Stirrer glands and bearings: These are used for the sealing of the stirrer shaft assembly and can be operated aseptically for a long duration. Four basic types of seal assembly have been used: The stuffing box(packed gland seal) The simple bush seal The mechanical seal Magnetic drive 3) Buffers: Used to prevent vortex and to improve aeration efficiency. Are metal strippers roughly 1/10th of the vessel diameter and are attached radially to the wall Normally 4 baffles are used, but in vessel over 3dm3 diameter 6-8 baffles may be used.

4) Sparger: Used for introducing air into the liquid in a fermenter. Three basic types of spargers are used: 1) Porous sparger 2) Orifice sparger 3) Nozzle sparger. Measurement and control system: Speed Control: Peed control relies on the feedback form tachometer located with drive motor determining the power delivered by the speed controller to maintain the speed set point valve set by the user. A digital display shows the actual speed in rpm, as determined by the tachometer signal. Temperature Control: Pt-100 temperature sensor is used to give an accurate indication of vessel temperature by relating changes in electrical resistance of the sensor to temperature. Temperature control is either by direct heating using a heater pad ir by circulating warm water around the vessel jacket. A cold finger is used to cool the vessel contents. pH control: It is achieved by the addition of either acid or alkali to correct changes in the pH of the culture during growth. The controller use a pH electrode (either glass or gel type) to ph changes provides a feedback signal. Control of dissolved oxygen: Dissolved oxygen is one of the most important yet one of the most difficult parameter to control properly during fermentation. The electrodes used to measure dissolved oxygen are of two types. Galvanic electrode which are generally simple, cheap, slow and have a limited life. Polarographic electrode which are generally more complex, accurate, rapid and robust but expensive. Antifoam control: The control foam is based on its detection by a conductance probe in the vessel head space, which leads to the controller delivering a dose of liquid antifoam reagents via a peristaltic pump, antifoam reagents can be material oils, vegetable oils or alcohol.

Measurements Agitator speed Agitator power Temperature

Flow rate

Dissolve oxygen pH Foam Redox Turbidity Pressure Liquid feed rate

Methods Frequency counter techogenerator Torque sensor. Electrical power Resistance Thermometer Thermistor Thermocouple Rota meter Orifice meter Thermal mass flow meter Galvanic probe Polarographic probe pH electrode Conductivity probe Redox electrode Turbidity sensor Pressure transducer Peristaltic pump Syringe pump Magnet flow meter

Comments More precise less reliable. Difficult Recommended. Probably best Fragile Satisfactory Not recommended Satisfactory Less accurate Set- point control Widely used Widely used Widely used Widely used Empirical valve complex Satisfactory Widely used Limited capacity Large

Result: Thus the fermenter parts, construction & control system have studied.

BATCH REACTOR
AIM: To run a batch reactor, analyse the critical parameters and measure the specific growth rate. Materials and Method: BASIC COMPONENTS: Medium Host Plasmid Glucose Concentration Inoculum O.D Ampicillin Nacl M9 MEDIUM PREPARATION: 1. Make M9 salts 2. To make M9 Salts aliquot 800ml H2O and add 64g Na2HPO4-7H2O 15g KH2PO4 2.5g Nacl 5.0g NH4cl Stir until dissolved Adjust to 1000ml with distilled H2O Sterilize by autoclaving 3. Measure ~ 700ml of distilled H2O (sterile) 4. Add 200ml of M9 salts 5. Add 2ml of 1M MgSO4 (sterile) 6. Add 20ml of 20% glucose (or other carbon source) 7. Add 100 l of 1M Cacl2 (sterile) 8. Adjust to 1000ml with distilled H2O PLASMID pTYB11 FEATURES: Total sequence length : 7212 bp 140-1000 140-208 1042-1555 1666-2254 beta-lactamase (bla; amp-r) CDS (start 140) beta-lactamase signal peptide CDS (start 140) M13 origin of replication (-+) pMB1 origin of replication (clockwise)

: M9 : E.coli GJ1158 : pTYB11 : 5g/L (initial) : 0.8 : 100 g/ml : 200mM

4840-4857 4859-4883 6503-6556

(RNAII - 35 to RNA/DNA switch point) T7promoter (transcript start 4840 clockwise) lac operator multiple cloning site (SapI-Smal)

REACTOR PARAMETERS: Total volume Reactor working volume Aeration pH Dissolved Oxygen Temperature

: 1.9L : 1/5L : 2L/min (initial) : 6.6 : 73% : 37oC

THEORY: Batch culture systems represent growth in a closed system. They use a flask or fermenter containing a suitable growth-supporting medium operated under optimum condition of temperature, pH, and redox potential. The medium is inoculated with the cells growth until some essential components of the medium is exhausted or the environment changes because of the accumulation of a toxic products, pH changes etc. In general microbial growth is determined by cell dry weight measurement or absorbance measurement. The growth curve can be divided into 3 phase: 1. Lag phase: In this phase, the cell adapts to the new environment by synthesizing necessary enzymes for the utilization of available substrates. 2. Exponential phase: The cell constituents in this phase increase at a constant rate. 3. Stationary phase: Cell death occurs because of depletion of essential nutrients but is balanced by cell growth hence there is no net growth or increase in cell number. This is followed by death phase. Specific growth rate: dX/dt = X Where is specific growth rate/ X is the biomass concentration On integration we ge, X = Xoeut, where Xo is the inoculum = (ln x2 - ln x2)/(t2 - t1) = (ln O.D2 - lnO.D1)/(t2 -t1) After inoculation, the parameters were periodically noted and tabulated. The biomass was tracked by measuring the O.D using UV-Visible spectrophotometer. The culture was induced to synthesize product by NaCl induction after 3 hours of running. Here the product of interest is GMCS (Granulocyte Macrophage Colony Stimulating Factor), an anticancer biological. Total batch run time = 6 hours.

Observations: S.No

Time (hr)

O.D

D.o%

Aeration(lpm)