STEAMING FERMENTORS

The use of hot and pressurized steam to clean and sterilize fermentors is one of the most crucial steps in the fermentation process. Yet how much do we really know about the hot steam and its effects and limitations on the fermentation process?

Steam is the gas phase of water. Steam is produced when water passes its boiling point. Thus the temperature of steam is always very hot. It is possible to increase the temperature of the steam by generating steam under pressure such as those produced during autoclaving. By manipulating the pressure and controlling the temperature of the steam produced it enhances the value of steam as a sterilizing agent. This makes steam as a very useful method or technology in cleaning and sterilizing fermentors.

As a very hot gas phase, steam is most suitable in cleansing and sterilizing various components of the fermentors as well as in CIP and COP operations. Its power to sterilize and clean can even go beyond the fermentors but the plant environment such as other utensils and even floorings and processing machineries

The advantage of using steam is that it has the ability to penetrate the most hidden and inaccessible places

USING STEAM TO STERILIZE

Using steam to sterilize the fermentor depends more on the size of the fermentors

For small size fermentors the normal autoclave and the standard operating sterilization parameters apply. But for larger fermentors at the level of pilot or industrial fermentors there need to be adjustments in the sterilizing parameters

Steam sterilization of pilot and industrial fermentors is a bit more complicated as CIP needs to be applied. The fermentor and feed-system piping must be steam sterilized.

The accepted procedure involves heating the entire system with steam to a minimum of 121°C for 60 minutes or more. In case of very large fermentor installations large industrial fermentation systems often prove more difficult to sterilize. Steam heating to temperatures approaching 150°C for sterilization periods approaching 4 hours are often needed to insure that all hard-to-reach internal areas have been brought to minimum sterilization temperatures.

The efficacy of the operating regimes for steam sterilization has to be individually determined and validated for each type of fermentation

In fermentation technology we are often faced with the choice of two kinds of steam; the saturated or wet steam and the dry steam. The "dry steam" describes a system that produces a high temperature with "low moisture vapor". This vapor contains only 5-6% water and is much less dense than the air we breathe.

The trick to get a dry can of steam is to know the volume of your can and the amount of H20 in the can. Assuming the can to be a constant volume and that the H2O cannot escape, you pick the temperature and pressure needed to get "dry" steam

Not many using the autoclaves know the difference between the terms psi and psig. Find out….!

We can use pressurized hot steam to sterilize the vessels without using autoclaves. However the temperature of the steam would not be as high as in the autoclaving process. There are a number of mobile steamers in the market that can provide in situ hot steam. This method of sterilizing would be ideal in cottage fermentation industries

Read more!

MICROBIAL CONTAINMENT

When we are dealing with microbial fermentation, we are really dealing with the growth of large number microorganisms in the fermentor. In such situations we are usually faced with two main problems:

1 Preventing the entry of unwanted microorganisms as microbial contaminants into the fermentor which can disrupt the fermentation process. In this situation we are referring to monoseptic fermentation and cultivation of animal and plant cells
2 Preventing the escape of the microorganisms from the internal environment of the fermentor into the surrounding environment. The escape of the microorganisms may occur through process failures or unintentionally. Although in most cases such situation may not constitute any risk as it does not involve pathogenic microorganisms or genetically engineered microorganisms. However, there is still risk which might arise due to the high concentrations of microorganisms released especially in areas in the vicinity of the fermentor or the plant

In the first situation, the prevention of unwanted microorganisms entering the fermentor is often achieved by the process of sterilization and maintenance of aseptic integrity of the fermentation system throughout the period of fermentation.

In the second situation, it is more the prevention of the escape of microorganisms or its destruction of microorganisms released from the fermentor. The released microorganisms do not affect the fermentation process but constitute a safety and health hazard. In the first situation the invasion of unwanted microorganisms will affect the fermentation process rather than being a hazard

In the analyses of microbial containment we must accept the following facts:

1 There is a very high concentration of microorganisms in the fermentor
2 What ever goes into the fermentor must come out, example air, fermentation medium
3 The fermentor is a high pressure vessel

DANGER OF AEROSOLS
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The highest threat in the microbial containment is the air line. Air that enters the fermentor will first have to pass through the fermentation broth containing the billions and billions of microorganisms before escaping through the head space and through the exhaust port. The volume of air passing through the fermentor depend on the pressure and volume of fermentor. Generally about 0.5 to 2 vvm of air is delivered through the fermentor. This will represent a very high volume of air that enters and leave the fermentor throughout the fermentation run all with the potential of carrying along billions of microbes to be released to the environment if not contained

The most common threat of spread of microorganisms from the fermentor is due to aerosols generated. Accidental release of aerosols could result in the widespread transportation and dispersal of microorganisms to the environment by air. Aerosols are easily generated in fermentors due to the presence of surface active compounds, pressurized air and high turbulence.

The existence of aerosols prolonged the survival and spread of the microorganisms due to the mobility of the aerosols and sustenance of the microbes through nutrients present in the aerosols and lower risks of dessication

DANGER OF SPILLAGE AND SPENT MEDIA
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The substrate for the fermentation and the fermentation broth left in the fermentor after termination of the fermentation contains high concentration of microorganisms and raw nutrients which are still able to support the microorganisms as well as microbial contaminants.
Given time and right temperature these microorganisms could proliferate to become a threat to the environment.

It is important that immediately at the conclusion of the fermentation after the fermentation broth is removed for downstream processing that washing,sterilizations be carried out immediately. This will prevent proliferation of the microbes and secondary problems arising from cross contaminations later on

TYPES OF MICROBIAL CONTAINMENT
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There are generally three types of microbial containment in any fermentation facility

1Primary containment
2Secondary containment
3Tertiary containment

Primary containment are activities carried out in the containment of the microbes at the level of the fermentor or bioreactor
Secondary containment are activities carried out at the level of the operator such as protective clothings
Tertiary containment are activities carried out at the level of the laboratory or plant facilities

The level of containment is the reflection of the status of biohazard of the facilities. Those laboratories dealing with very dangerous microorganisms will need the highest level of containment at primary, secondary and tertiary containment level.

KILLING THE MICROBES
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Air or the exhaust gas coming out of the fermentor is the main point of containment control. There are two main approaches to it:

1 Sterilization by filters
2 Killing by heat incineration or disinfectants

Before opting to which choice you will take, one have first of all to consider the physical, biochemical and microbiological characteristics of the exhaust air. Technical and economical constraints to have to be considered as well as the level of biohazards faced



PROTOCOL OF MAINTENANCE OF CONTAINMENT
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Should cover the following:
1 Environmental monitoring
2 Work practices- process protocol, hygiene requirements and clothing, emergency procedures
3 Medical surveillance
4 Worker education and training
5 Engineering controls- physical containment, exhaust gas control,, ventilation
6 Validation of functionality of containment facilities- complete sterilization.physical containment and fermentation termination Read more!

POST STERILIZATION ACTIVITIES

Remove the fermentor gradually with care while the temperature is still warm and tolerable

Add air line with sterilized air to maintain positive pressure within the fermentor. Condensation of steam might lead to negative pressure and suction of contaminants from the surrounding

Use a slow stirrer speed to improve heat loss from fermentor

Leave fermentor standing till its cool enough to be inoculated if not the culture will die. Possible leave the cooling of fermentor in aseptic area or inoculation chamber Read more!

EFFECTS OF AUTOCLAVING ON FERMENTORS

Most fermentors are sterilized by autoclaving, or hot steam under pressure. For small laboratory fermentors they are sterilized in autoclaves. In the case of large fermentors, most if not all are equipped with in situ sterilization facilities built into the fermentor system.

For most autoclaving sterilizations for both small and large fermentors, the accepted autoclaving conditions is at 121 degrees Centigrade at pressure of 15 to 20 psi. The autoclaving holding time about 15 to 20 minutes.

For very large industrial fermentors, depending on various other factors as loading and target kill, the sterilization regimes could be different or individually determined.

During sterilizations by autoclavings, the fermentors and its contents are subjected to very high temperature, pressure and long contact time. The fermentors undertaking the sterilization will be subjected to a high degree of physical abuse and the reactions of the fermentor could be as follows

1 There will be expansions in the volume of the fermentor as materials expand under high temperature. The fermentor will face additional problems as the structure of the fermentor is made up of various materials such as glass, steel, rubber etc. Each of the component material will show different coefficient of expansions

2 High heat will cause water to evaporate. So it is not surprising that there will be a loss of water from the fermentation broth by a few percent. The loss of water will either result in:

a) the broth being more concentrated or
b) there will be an increase in the surface area to volume ration of the content of the broth leading to faster evaporation rates

In making up for the loss of water additional water has to be added to compensate for the potential loss of water by autoclaving

3 There will be a degree of nutrient degradation especially for sensitive labile organic compounds

4 There will be unwanted side reactions such as between sugars and amines leading to the toxic Maillard's reactions

TOWARDS PROPER AUTOCLAVING OF FERMENTORS
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An autoclave in simplicity is just a more complex pressure cooker. To be efficient in autoclaving it is very important to drive off any air pockets that might be present in the autoclave. Air is a poor heat conductor. If the air is not driven out it will be difficult to bring the right temperature in all the autoclave. Let the autocleave heat and steam up and release the hot steam through an escape valve before closing the valve and starting the sterilization process

Ensure that the temperature recorded in the autoclave chamber is uniform throughout the whole chamber. Make sure the temperature stated on the panel outside the autoclave is the real temperature inside the autoclave chamber. We do not want under heating and over heating to occur. Place thermo probes to measure the real temperature of the autoclave and repair if needed

Do not overload the autoclaving chamber. This might lead to poor degree of sterilization being achieved.

Ensure that the autoclave is not leaking or suffering from leak in pressure as it will affect the sterilization process Read more!

PREPARING THE FERMENTOR FOR STERILIZATIONS

This discussion will be using a laboratory scale fermentor as the example.

After the fermentor vessel has been thoroughly cleaned, rinsed and dried, it is ready to be prepared for sterilization in the autoclave. The following main steps should be taken:

(NOTE: DURING THESE STEPS EXTRA CAUTION MUST BE TAKEN TO AVOID DAMAGES)

1The vessel seals should be removed and lightly greased with silicone grease
2Fill up the vessel with the desired fermentation medium. Ensure that about a headspace of
about 20% of the vessel volume be retained
3Replaced the top plate onto the vessel and clamp and tighten all bolts
4 Insert the relevant electrodes
5 Do the same for all other tubing connections
6 All electrodes terminals capped to avoid water
7 Ensure one opening filtered to allow air to escape

DETAILS OF THE ABOVE STEPS
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Normally lab scale fermentors are small vessels which have a glass body with a glass or stainless steel top plates. Since we are dealing with glass, the risk of the fermentor being accidently mishandled and broken is high. This even applies especially to the glass ph electrodes which usually comes with a glass casing.

Be sure to handle the glass body tightly and properly. Rest the glass body slowly and carefully. Make sure the glass body is rested or stand in a very stable position so as not to roll or move accidently

The glass electrodes must be placed in a special tray with soft padding to be easily transported around. A clean tray should also be used to keep all the nuts, screws, clips, tubings, filters that belong to the fermentor in questioned.

Ensure that silicone grease is used to seal the various O-rings. This grease is suitable because it can withstand high temperature and vacuum. Make sure not to over grease as it may create problems of fittings bungs or rubber stoppers (if used)

When filling the fermentor with the fermentation media, it is more convenient to do it before placing the top plate. After filling to the desired volume, place the top plate onto the body of the fermentor.

Tighten the bolts and nuts slowly and gently. Ensure opposing nuts are tightened simultaneously. Do not over tighten the nut or blocker because it will damage the fermenter threads and the O- rings

Insert the relevant electrodes slowly and properly aligned. It is better to wet the e;ectrodes to improve the lubrication and make the insertion easier. At the end of inserting the electrode tighten the nuts slowly and gently

There are various type of tubing sizes to be used in setting up the fermentor. Ensure the tubings used are of tygon type or silicone rubber. If older tubings are to be used ensure that its not brittle or damaged. Cut of the tips of damaged tubings before making connections. Ensure that the ends of the tubings are clipped properly to maintain aseptic integrity

Tubings used for water jacket should be pressure tubings. Do not lock up the male nut into the female nuts as it might lead to high pressure developing during sterilization leading to breakage

Cap or cover all terminal ends or port holes ends with aluminium to prevent wetting

LAST CHECKING
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This is the most important final step before the fermentor enters the autoclave. Ensure all outlets are tightened
Ensure there is one port hole or outlet opened to let air escape
Ensure all tubes or insertions correctly positioned

...and may God bless you! Read more!

HAZARDS IN A FERMENTATION LABORATORY OR PLANT

Have anyone of you tried visiting a fermentation factory or plant? If you have you will know the experience....

Huge fermentors that stretch from the floor almost to the ceiling
High over head gantries
Humid and warm environment
Noisy with all the steam hissing sounds
Motors everywhere

Sometimes it looks as if these fermentation laboratories or plants are located in workshops, with tools and skids everywhere..

It is a dangerous and hazardous place to be and safety precautions are of the utmost. Accidents do easily happen here if no precautions are taken.

The type of hazards that might occur can be classified into the following areas:

1 Microbiological hazards
2 Physical hazards
3 Chemical hazards
4 Electrical hazards

MICROBIOLOGICAL HAZARDS
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In the fermentation industries or laboratories, we are dealing and always exposed to very high concentrations of microorganisms. If taken care and proper control these high concentration microorganisms are always contained in the bioreactor where they are purpose grown.

Depending on the type of fermentation process, the microorganisms used may or may not be pathogenic. Any danger of release of pathogenic microorganisms would be a real threat to the surroundings

The risk of mirobial hazards can occur at all stages of the fermentation activity right up from upstream, mid stream and down stream. Of course the most crucial stage will be the period the microorganisms are grown in the fermentor. There are billions and billions of microorganisms "swimming" in the fermentation broth!

Microorganisms can only escape due to poor handling procedures that is poor microbiological techniques. They can also be released by accidental discharge or poor containment of the microorganisms in the fermentor

In aerobic fermentors, air or oxygen is actively supplied into the broth of the fermentor for the use of the aerobic microorganisms. Most of the air will be released through the exhaust outlet to the environment. If the exhaust air is not sterilized or filtered, there is the danger that aerosols carrying the microorganisms from the fermentor will be released.

The fermentor is usually operated skightly under high pressure of about two bars. Any sudden release of the pressure through any of the valves risk releasing and spreading the microbes.

The presence of aerosols released from the pressured fermentor will exacerbate the problem of microbial dispersion and transmission. Not only will the aerosols be the " carrier" for the microbes but it will even protect the microbes from premature dessication and even provide nutrients for the microbes

Microbial containment should be of the highest order in managing pathogenic bacterial and viral fermentation

CHEMICAL HAZARDS
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There are a few possible chemical hazards that may arise in operating the fermentor or in the fermentation plant. Chemicals that may be used in the fermentation plant are from a few sources"

1 Calibration gases
2 Gases used in fermentation such as nitrogen, oxygen, hydrogen, carbon dioxide, NH3 gas
3 Solvents such as alcohols
4 Surfactants
5 Chemicals used as substrates
6 Acids and alkalies
7 Disinfectants

Some of these chemicals are toxic, flammable, corrosive and may be dangerous if not used properly

PHYSICAL HAZARDS
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Physical dangers that lurked in the fermentation laboratory or plant may include:
1 Heat
2 Pressure
3 Slips and falls
4 Knocks
5 Fire and burn hazards
6 Cuts and bruises

Dangers from high heat source occur in such activities as sterilizing the fermentors or from the use of autoclaves.
High pressure accidents too can occur by improper use of fermentors and autoclaves

Slippery fermentation plant floors could easily lead to slips and injuries. Cuts and bruises are common during handling of the fermentor

Equipments and tools left haphazardly could lead to unwanted accidents and falls

Improper use of flaming during aseptic procedures could lead to accidental burnings and personal injuries

ELECTRICAL HAZARDS
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Electrical accidents such as electric shocks is common in any fermentation laboratory. Short circuits are common. High voltage and high amps current are often used regularly in the fermentation operation

Fermentation operatives should be trained to identify all the possible danger points and steps taken should accidents happen. This will be discussed in future blogs Read more!