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6.1 Introduction

When considering the setting up of a laboratory for fermentation, a number of points should be kept in mind.

Scale of operation. Clearly, at the bench-top scale (up to 3 litres volume), little additional adaptation would be required in a standard microbiology laboratory. Larger-volume fermenters (usually mounted on skids, trolleys, or frames) require more extensive preplanning of the operational area. Such fermenters, room 5-50 litres working volume, might well be termed research scale; anything larger would come into category of a small pilot plant. Small scale processing is from 50 to several hundred litres working volume.

Type of fermenters. Type of reactor, used at every operational scale, has a much higher degree of operational flexibility then most other fermenters.

Number of fermenters. The use of one or two bench fermenters will require only a source of electric power, water, and access to a small autoclave. Operation of a number of large vessels will have considerable impact on lab design; for example, it becomes worthwhile arranging steam line to each fermenter point to allow for in situ sterilization.

Nature of fermentation process. This really relates to the type of organism to be used; the use of fungi will require equipment quite different in some respects from that employed for "traditional" microbial fermentation such as Bacillus thuringiensis.

In the description of a fermentation laboratory which follows, it should be remembered that this describes the "ideal", a lab constructed ab finite for the purposes of microbial fermentation. Such an "ideal" is rarely achieved, but is always a useful target.

6.2 Pilot Plant

A fermentation laboratory is primarily a microbiology laboratory incorporating advanced technology equipment, and as such should be designed to maximize efficiency while maintaining a high safety standard. The general layout of the laboratory will depend on the shape and size of the room allocated, and it is advisable for laboratory personnel and scale production.

The laboratory is divided into two or three distinct areas:

(i) Wet floor area. This houses all fermenters, stills, autoclaves, centrifuge.
(ii) General laboratory area. Used for medium preparation and basic "wet" processes, i.e. dry weights, viscosity, pH, and measurements
(iii) Formulation area.

A fermentation laboratory has two distinct areas, the wet floor and the dry floor.

All fermenters, including bench top varieties, should be situated on the wet floor area so that spillages, intentional or not, can do no damage either in the laboratory or to people and equipment situated on the floor below. Wet floor should be constructed to meet two main criteria. it should be non-slip, whether wet or dry, and should be easily cleaned. These factors are often not compatible, i.e. the rough surface required to make the floor non-slip is often ideal for harbouring dirt. The wet floor should be constructed so that it slants towards a central drainage channel (fermenters should be levelled using adjustable feet or blocks); the channel itself should slant towards the drainage output of the laboratory, and must be covered by a stainless steel or heavy-duty plastic grid to prevent accidents.

The dry floor area of the laboratory should be covered with seamless sheet vinyl which has a smooth surface and does not catch dirt.

The junction between the wet and dry floor areas of the laboratory is an area of potential danger, as sheet vinyl is slippy when wet. It is advisable to introduce an area where shoes can be dried between the two floor types.

Routine cleaning of both floor areas is essential. A wide range of detergents/disinfectant is available, and the product chosen will depend on the biocidal effectiveness required. Because of the nature of equipment and fermenter contents, trained personnel only should clean the wet floor.

6.3 Laboratory Equipment

These pieces of glassware can vary in size and form and in some instances have been designed and developed for specialist application.

Shaker tables were designed to assist with oxygen transfer. These tables are designed to run for long periods of time and be free from vibration. The tables are driven by a motor, and normally a rotary shaking action or reciprocating shaking action is produced.

These shakers have to be robust and reliable with no vibration and silent running conditions. One can have a more sophisticated shaker by having an incubator shaking cabinet for shake-flask fermentation in a precisely defined environment. These cabinets can control the temperature, illumination, gaseous levels, and humidity.

Increasing the speed of a shaker can increase the oxygen transfer rate of a particular flask, therefore the optimum speed for that flask and culture has to be found by trial and error.

The lower the volume of medium in a shake flask, the better will be the OTR (Oxygen Transport Rate). The minimum volume that can be practically obtained (e.g. 50 ml in a 250 ml shake flask) should give the best OTR and hence the best results. This will also be dependent on sample volume. Very low volumes can only be used for short-term fermentations, otherwise the medium will evaporate and the nutrients would become too concentrated for the culture to perform satisfactorily.

The standard 250 ml Erlenmeyer flask is cheap and simple; most of the shaker tables designed to use these flasks although there are tables which can be adapted to allow other shapes or bigger flasks.

Baffles have been used in shake flasks to assist in the OTR, as well as preventing vortex formation, but there are only really suitable for low-volume short-term fermentations because of splashing which leads to the cotton-wool plug becoming damp preventing free flow oxygen.

Different plugs can be made of cotton-wool, glass wool, polyurethane foam, gauze or synthetic fibrous material. (An aluminium foil cup can sometimes be used in conjunction with these plugs). The plug has to be prevent airborne microorganisms from getting into the medium while at the same time allowing free flow of air into the flask, and for this reason it must not be allowed to become wet.

Basically, the stirred fermenter consists of a cylindrical tube a top-driven or bottom-driven agitator. The stirred fermenter with a top-drive assembly is the most commonly used fermenter because of its ease of operation, neat design, reliability, and robustness.

For smaller laboratory fermenters (bench-top), borosilicate glass is used as the cylindrical tank and a top plate of stainless steel clamped on. A motor is fixed above the top plate and is attached to the shaft. The motor can be uncoupled. The vessel, medium and probes are usually sterilized together in an autoclave, and minimizing the number of aseptic operations required (Fig. 6-2).

These glass vessels can vary in size from one liter to 20 liter capacity. The vessel itself will have a specific impeller design, baffles, an air sparger, and sample port.

The special "Rolls Royce" laboratory fermenter is constructed like hollow steel cylinder with either top or bottom drive and can be cleaned and sterilized in situ. These stirred fermenters can vary in volume from one liter to 100 litres capacity. Obviously they are more expensive than the glass vessels but they are more robust, reliable, and designed to a lifetime.

Often large samples or regular samples have to be taken for analysis during the fermentation. These volumes must be take into consideration when choosing a fermenter.

Bench-top fermenters are usually cheaper to purchase than the trolley- mounted or skid mounted fermenters. This is partly due to the fact that their instrumentation is often not as sophisticated as a laboratory or research fermenters. This latter have a sophisticated instrument control package for pH, temperature, and agitation, and this obviously costs more.

Most stainless steel fermenters are designed with bottom drive unit which is belt driven. This has several advantages:

It allows easy access to the top of the vessel and all the moving parts can be isolated and encased underneath the vessel, thus making it a safer piece of apparatus;

With the motor belt driving the agitator any spillage which occur will not fall onto the motor because it is not sited directly below the agitator shaft.

The agitation shaft should have a double mechanical seal which ensures that the medium does not leak out at the shaft housing. The agitation shaft will normally have two or thee impellers, each with four or six blades depending upon mixing requirements.

Membrane type filters, containing a cellulose acetate, or nitrate membrane of known and consistent pore size, which therefore retains all particles larger than that pore size 0.2 mm or 0.45 æm pore size filters are suitable for most applications. These filters are relatively cheap, disposable, and usually readily inspected for blocking or fouling. They should be discarded after a fixed number of autoclave cycles. The manufacturer will normally indicate the number of cycles which can be withstood.

Packed-bed type filters, these filter have no uniform pore size and the mechanism of particle removal tends to be rather more complex. Typically, a filter housing is packed with glass wool or non-absorbent cotton wool. Such filters are vulnerable to compaction and to wetting which may allow channelling to occur. Sudden fluctuations in the pressure drop across the filter can cause release of particles or packing material under some circumstances. Such filters are readily constructed in-house, but their sole advantage is cheapness.

Cartridge filters are composed of a stainless steel or polycarbonate filter housing containing a removable filter element. The filter element is often composed of a hydrophobic material (e.g. PTFE) bonded in polypropylene. These filters can be steam sterilized in situ or autoclaved. They are initially more expensive than other types, but their reliability and durability make up for this.

The sterile air is than fed into the bottom of the fermenter dispersed by a sparger and thoroughly mixed into the medium by the agitation system.

Vent gases can be filtered by the same means, but one has to be aware of the risks of blockage due to carry-over of medium or foam-out. Such risks can be minimized by the use of a foam control system; either a mechanical system (offered as an option by some fermenter manufactures) or, more routinely, a system involving addition of some foam-suppressing chemicals (e.g. a silicone-based compound or polypropylene glycol). An efficient condenser fitted to the gas outlet will also reduce the likelihood of exit filter blockage.

A number of essential services are required to run an efficient fermentation laboratory, namely air, steam, water, electricity, and if it is possible the gas. The level of each service required depends on the size of laboratory and the demands on that service. All services should be available 24 hours a day, seven days a week, for a dedicated fermentation laboratory; smaller laboratories housing only a small number of simple fermenters such as polyethylene cushions fermenter do not require this level of service.

Air is required for many purposes in a fermentation laboratory, e.g. aeration of fermenters, operation of hydraulic autoclave doors, and calibration of gas analysis equipment.

Laboratory fermenters are often supplied with integral air pumps. Individual air pumps can be purchased to supply air vessels which do not have an integral air supply and which are situated in laboratories without service air. The ideal pump is the diaphragm pump. These pumps vary greatly in their capacity and should be purchased according to the demand of the vessel. It is essential that the pumps be oil free and be suitable for prolonged continuous usage. Diaphragm pumps are relatively cheap and are useful if only one or two vessels require an air supply.

For larger laboratories, a more sophisticated air supply is required and a compressor should be purchased. The type and size of compressor selected depends on the function it is to have in laboratory. The compressor should be able to meet the current demands of the laboratory with additional capacity to allow subsequent growth.

Fermentation processes require particle-free clean air that meets food grade standards, so the compressors utilized must be oil-free units. Beware of compressors which are claimed to produce oil-free air; these are not oil-free compressors and should be avoided since even when functioning well they can allow oil to escape into the system, leading to fouling of lines and contamination of the fermentation. If such a system is already installed, the air lines must have reelable protective filters downstream of the compressor which are regularly cleaned and serviced.

To ensure a constant supply of air at all times it is advisable to operate two compressors so that if one fails for any reason, the other unit will cut in and maintain the supply going. It is necessary to ensure that each compressor is used on alternate days so that the demand on each machine is equal. Incorporating an air receiver in the supply line decreases the demand on the compressor and also allows condensate to be removed from the supply.

If the compressor is to be situated in the laboratory, acoustic hoods should be fitted to reduce the noise level. Ideally the compressor(s) will be housed in a room situated adjacent to the fermentation laboratory.

The air supply is normally taken from the compressor to the fermenter via network of pipes. The pipework should be made of a non-corrosive material with a smooth interior to prevent build-up of dirt. Half-inch steel piping is excellent for this purpose and is also relative cheap. Pressure-reducing valves must, therefore, be installed downstream of the compressor along with oil and water traps (as compressed air expands in the lines a small amount of water is formed 0.025 g H₂O per liter air at 25^oC). All reducers and traps should be regularly cleaned and serviced. All connections made in the lines must be able to withstand to pressure at which the air is to be delivered (frequently 1-2 bar g. but this depends on the fermenter(s) used). It is, therefore, necessary to have the systems of pipework installed by qualified plumber.

Steam is required in a fermentation laboratory for sterilizing fermenters, controlling temperature in large vessels, autoclaving, and supplying steamers. Each fermenter be should have its own steam supply which can be isolated from the vessel by means of a gate valve. The steam supply should be as dry as possible, and all lines in the laboratory should be well lagged to help prevent the formation of condensate. Lagging is also important for the protection of laboratory personnel. The lagging should be covered with an aluminium casing to give a finish that looks more presentable, is easily cleaned and prevents the lagging material getting damp.

As with the air supply it may be necessary to incorporate pressure reducers in the lines. These should be carried out by qualified personnel.

A fermentation laboratory requires a constant supply of water to service fermenters, downstream processing equipment, and autoclaves, as well as for analytical purposes.

Mains water is normally connected to the service inlet on the fermenter chassis. Manufacturers will indicate the maximum inlet pressure a particular model of fermenter can cope with. This is often very different for different vessels, e.g. a small lab fermenter is generally serviced by water supplied at 1 bar g whereas a small-scale vessel may required up to 3 bar g. The mains water inlet pressure must be sufficient to meet the demands of the laboratory; if necessary, pressure reduction valves can be fitted on line to each vessel to allow a range of a different demands to be serviced.

The laboratory must also be supplied with good-quality water, for medium preparation and in areas where the local supply water is hard it may be necessary to install a deionizing system.

Water is also required for the basic laboratory chores of washing glassware, etc., and hand washing.

Electricity is required for lighting and for supplying power to complex array of machinery and instrumentation found in a laboratory.

Many items of equipment in a fermentation laboratory draw amounts of power, e.g. small scale vessels, autoclaves, and downstream processing equipment. It is, therefore, necessary in dedicated fermentation laboratories to have a supply of three phase electricity. Installation of three phase supplies can be expensive; it is advisable, therefore, to have a supply in the laboratory and to take off lines as and when required.

Every fermenter in the laboratory will require a number of electrical sockets supplying power to fermenter, pump and additional equipment, such as portable pH and O₂ meters. A bank of six sockets per fermenter is ideal for this purpose. For larger fermenters requiring a three-phase supply it is still very useful to have a bank of single phase sockets which should be installed alongside the three-phase supply.

Natural gas should be supplied to the general laboratory area usage. Speciality gases, e.g. carbon dioxide, oxygen, and calibration gases, are usually provided in the form of compressed gas cylinders which should be secured with safety straps.

6.4 Fermenters

A well-equipped fermentation laboratory requires substantial capital investment for the purchase of fermenters and a wide range of ancillary equipment (see ANNEX 2.).

6.5 Ancillary Equipment

The size and type of autoclave purchased will depend on the number of fermenters being serviced. A standard size autoclave, e.g. 450 x 800 x 1200 mm chamber dimension, will be sufficient to deal with ancillary purposes, e.g. sterilizing glassware, or preparation of small quantities of media.

Bench-top or portable autoclaves are useful for sterilizing small items, e.g. bottles, a small number of 500 ml conical flasks. Pressure cookers can be used for this purpose. but the life span of the portable autoclave is general longer, because it is more robust in construction.

Standard Autoclaves small volumes from 1 to 10 litres

Incubators are required for the cultivation of stock cultures and production of inocula.

Two types of oven are useful in fermentation

(i) Hot air ovens can be used for drying glassware or dry weights and sterilizing Ovens.
(ii) Microwave Ovens are used for dry weights, drying glassware, melting agar etc.

At laboratory scale, liquid pump is achieved almost exclusively by means of peristaltic pumps. The choice of pumps will depend on the application.

These fixed-speed pumps are normally used for addition of acid/alkali, antifoam linked to a pH/antifoam controller.

Lager pumps, used for example for nutrient addition in continuous or fed-batch culture, generally have clamps to hold tubing on the pump heads. Such pumps, which take tubing of up to 8 mm bore, can be either fixed speed or variable speed, manual or auto control.

If is often necessary to sterilize larger volumes of medium separately from the fermentation vessel, then pump the sterile medium into the pre-sterilized vessel. For such purposes, a high-speed pump, capable of delivering at least 3-5 liter min-1 using standard 5-6 mm bore tubing, is very useful.

When buying a pump, it is important to think carefully about the intended flow rate range. Due to nature of electric motors, pumps are most accurate at high speeds and least accurate at very low settings. On the other hand, tube life is much reduced at high speeds.

6.6 Medium Reservoirs

Particularly in fed-batch and continuous culture it is essential to have a number of medium supply vessels. Such vessels must be able to withstand repeated autoclaving. The volume of vessels required (and the number) will depend on the experimental programme, e.g. a 10 litre fermenter operated at a dilution rate of 0,1 h^-1 will use 24 litres per day, so 5 litre reservoirs would be inappropriate. In practice, glass (heat resistant) and polycarbonate vessels are superior to glass because they are lighter, not so dangerous if dropped, and to show distinct signs of wear before they fall, whereas an undetected fault in a glass vessels can cause a sudden failure. After autoclaving, allow vessel and contents to cool before removing them. Medium components likely to be affected by heat should be filter sterilized and added aseptically later.

6.7 Cooling System

The medium after sterilization in the pilot plant fermenters must be cooling before inoculation during two or four hours.

6.8 "O" - Rings

O-rings are in constant use in a fermentation laboratory. They are used as the compressible material when a seal is made between glass and metal or between metal and metal. O-Rings are usually composed of nitrile or butyl rubber, sometimes of silicone. Remember that items such O-rings have a finite life span dependent upon how often they are autoclaved, and whether or not they are deformed by over-compression; they will thus have to be carried out as part of the regular maintenance programme.

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