(THE PICTURE TAKEN FROM WWW.SRC.SK.CA)
Economics is the single factor that dictate whether a particular industrial fermentation process is viable despite the fermentation process being technically viable. There is always the need to produce fermentation products at minimal costs so that the product will be cheaper on the market in the face of stiff competition from other producers
Various strategies are often taken to cut the production costs down such as:
1 Producing higher volume of the fermentation products using larger fermentors
2 Using cheaper source of fermentation raw materials
3 Cutting wastage of materials by using it efficiently
4 Making the fermentation process more efficient by using higher producing strains or better unit processes in the fermentation process
There are times however where the fermentation companies see a potential increase in demand for their fermentation products and decided that whether it is the best option to consider enlarging the capacity of their production by increasing the number and size of fermentors? This is a very risky decision to take as increasing the number and size of fermentors is a very huge investment in capital. Would it not be better if we can increase the efficiency of the existing fermentation process to its most achievable limit of optimization before deciding if an increase in production capacity is a viable option
While it is true that increasing the numbers and size of fermentors could increase the production capacity of the company, but if it is run at the same inefficiency as before there is going to be a high increase in the amount of potential wastes from operating costs to wasted fermentation media
The most important step therefore is to find ways of increasing the efficiency of the existing fermentation process and find at which step from the upstream activities to downstream activities where they are found to be rate limiting to the whole fermentation process. Proper analyses or studies must be carried out in terms time spent at each step.
TIME IS ESSENCE
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try shortening it at each unit until total efficiency objective is reached at each component step. This even include the CIP/COP times! Use a more efficient method of cleaning and more effective chemicals. This will allow a shorter turn around time for the fermentors to be reused again!
If every unit or steps in the fermentation process makes effort and improve their efficiency and time management then the overall fermentation process will be improved.
Meetings must be held regularly to see how the new fermentation run schedule could be further improved with the increase in efficiency and overall shortening of the time
Secondly, even the rate limiting steps are identified and adjusted, it might create different bottlenecks down stream to the rate limiting steps. Fermentation process is a dynamic process whereby any step changed upstream will affect the various downstream steps. There will also need to be changes to be carried out upstream to accommodate the different flow rate to the primary recovery step
ANALYSES OF THE RATE LIMITING STEP
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We can picture the industrial fermentation process involving the four main stages:
PRECULTURE --> FERMENTATION-->PRIMARY RECOVERY -->DOWNSTREAM PROCESSING
It is very obvious from the above flow chart that the most rate limiting step is the primary recovery step. Any change in the primary recovery step positively will also mean changes both upstream and down stream to the primary recovery step. Adjustments in both directions have to be made
FACILITATING THE FLOW
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In any industrial fermentation process could be seen as a series of events occurring or passing through a series of unit processes which end up as the 'packaged product' at the end. A good fermentation production floor is no better than a kitchen at home. A well designed kitchen equipped with the proper equipments and tools at the right place properly positioned will help improve the fermentation cooking process
The flow of the fermentation process must be maintained efficiently with no hold up and proper equipments in placed
Side activities that enter into the flow such as media preparation, sampling and analysis must enter the flow at the right time and place
Read more!
Thursday, January 31, 2008
STREAMLINING FERMENTATION- PROFIT OR EFFICIENCY?
Tuesday, January 29, 2008
CLEAN ROOM FERMENTATION
The new era of fermentation technology begins with the cultivation of mammalian, human, plant and microbial cells for the production of human biologics in the pharmaceutical industries. Traditionally before this, the type of fermentation are not that demanding or sensitive enough and are always carried out safely in large fermentors in various fermentation plants. This was and still the age of 'large volume and low value' fermentation industries.
With the pharmaceuticals now looking for newer and better way to produce biologics for the medical market, the trend is now towards producing 'low volume high value' fermentation products using single cell cultivations. There are reasons why such 'low volume and high value' fermentation could not be carried out as in the case of the traditional industrial fermentations, but this will not be discussed here at this stage
Modern pharmaceutical plants which include simple pharmaceuticals mixings, batchings and packings have always been carried in a very highly clean and sanitised environment free from the intrusion of most microbes in order to avoid contaminations of the pharmaceuticals produced. Any microbial contaminations which occur during the manufacturing of the various drugs will have very serious consequences on the state of health of the patients especially in the manufacture of parentrals drugs. Patients which are weak or with poor immune system will be easily compromised. The presence of the microbes during the manufacture and handling of the pharmaceuticals will easily lead to biodegradation of the drugs and affecting its potency.
It is therefore a standard requirement in most pharmaceutical manufacturing facilities that a very high grade cGMP is always adhered to in order to ensure freedom from microbes or the risks of microbial contaminants during its manufacturing process. Often the whole production floor is designed and built according to clean room specifications, especially at the most sensitive process areas such as the mixing or batching room and vial injection or filling suites.
This concept of pharmaceutical manufacturing takes a more critical turn when it involves the production of pharmaceuticals such as vaccines by the process of fermentation. The fermentation of pharmaceuticals is complicated by various factors such as:
1 Involvement of various unit processes at upstream, mid stream and down stream activities
2 Employment of heat, gases. water
3 High operator activities
4 High concentration of microorganisms or cells that could risk the environment and vice versa
CLEAN ROOM FERMENTATION
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A clean room could be defined simply as a air tight room where particles, temperature and humidity is controlled to prevent entry and maintained the level of cleanliness to a set standard
in order for production or research to be undertaken in these places, people, raw materials, products and production utilities (such as deionized water, gas, cooling water, exhaust air, drainage) have to be let in and out.
The design of the clean room should have the following facilities:
1 logical layout for the related facilities,
2 proper planning for circulation of people and objects,
3 proper barrier systems such as air showers, pass boxes and room air pressure control. These
barriers will prevent cross contaminations and maintain good cleanliness
SERVICE SECTION
This area of the clean room covers the following areas:
reception or receiving area,
quarantine area,
sampling area,
storage area
raw materials weighing area
cleaning, sterilisation and preparation of reusable small equipment area.FERMENTATION SECTION
The fermentation section houses the various equipments for:
1) media preparations which have several stainless steel stirred vessels,
2) fermentation which have a range of different sized fermentors,
3) cell harvest consisting of various filters and centrifuges,
4) cell lysis which houses several high pressure homogenizers
5) clarification-
DOWNSTREAM PROCESSING
This area includes equipments such as:
1) stainless steel stirred vessels for refolding,
2) buffer preparation
3) units for chromatography
Bulk filling laminar flow box ( class 100) in a class 100.000 cleanroom environment.
SEPARATE HEATING VENTILATION AIR CONDITION (HVAC ) SYSTEMS ----------------------------- All three cleanroom sections are controlled by separate HVAC systems.
A separate (HVAC) system provides a controlled, reproducible environment for the Clean Room as well as a comfortable environment for the people working in the facility.
The controlled environmental attributes include:
1temperature,
2air quality (HEPA filtration),
3 air change rates (ventilation),
4humidity, and
5pressurization relative to the individual rooms of the Clean Room and the outside air.
The HVAC system serves the following rooms:
The following class of particle removals are applied:
1 Preparation room Class 100000
2 Gowning Class 10 000
3 Batching or mixing room Class 1000
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The differential pressure values between the monitored rooms of the facility are as follows:
Batching vs gowning more or equal to 13 (Pa)
Gowning vs preparation room more or equal 13 (Pa)
SERVICES ------------ Important services for running the fermentors in the clean room environment such as clean steam, clean water and for Cleaning In Place are usually supplied through hard pipes from other areas such as basement or adjacent building. Separate pipe systems are provided for separate CIP systems.
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continueeeeeee Read more!
CONSIDERATIONS IN DESIGNING A FERMENTATION MEDIA
(PICTURE TAKEN FROM NATALIE94.VOX.COM)
The fermentation media or the feedstock represents one of the most expensive and important component in any industrial fermentation process. The fermentation media is the raw ingredients that are going to be used by the fermenting microorganisms in the fermentor and be transformed into the valuable fermentation products. In the process some of the fermentation media itself will be used by the fermentation microorganisms to be converted into energy and some of the nutrients in the fermentation media will be transformed into the formation of biomass or new microbial cells.
NUTRITIONAL REQUIREMENTS OF FERMENTATION MEDIA
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The fermentation feedstock is going to be the substrate that will support the growth of the microorganisms in the fermentation process. The first thing that should be considered in choosing or designing the fermentation stock is that the fermentation stock must contain all the nutrients the microorganisms need for its growth.
What type of nutrients the microorganisms need depends on:
1 The choice of the microorganism used in the fermentation
2 Physiological status of the microorganisms.
In certain fermentations, the nutrient requirements are simple but there are microorganisms and especially in the cultivation of mammalian cells where the nutrient requirements may be stringent. In certain fastidious microorganisms certain vitamins, growth factors may need to be added. In the cultivation of mammalian and plant cells certain serums or hormones may have to be added.
The physiological state of the microorganisms in the fermentation process is important. While high carbon might be needed during the log growth phase, the input of carbon have to be controlled during the stationary phase especially in the production of secondary metabolites
In general all microorganisms need the following classes of nutrients:
1 Primary elements
2 Secondary or trace elements
3 Growth factors
The type elements needed may vary with the type and physiological status pf the microorganisms
In searching for the suitable fermentation stock biochemical and nutritional analyses of the feedstock material need to be determined first before other factors are considered
THE FINAL OBJECTIVE IS THE FERMENTATION PRODUCT
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Despite saying that the feedstock support the growth of the fermentation microorganisms but in the eyes of the fermentation technologists they see only that :
FERMENTATION FEEDSTOCK===> FERMENTATION PRODUCTS
They are always looking for processes that gives the highest conversion rate from substrate to products. They are also looking at the costs of producing the fermentation products.
So in choosing the fermentation feedstock they will have to consider the cost of production if using certain choice of fermentation feedstock!
ECONOMIC CONSIDERATIONS
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As in a simple assumption that it is the fermentation media or feedstock that will be converted to the fermentation products, the feedstock must be :
1 Easily available in large volume
2 Low costs
It is common therefor in any industrial fermentation the source of fermentation feedstock arise from agricultural or industrial wastes.
CHOOSING THE CHOICE OF WASTES AS FERMENTATION FEED
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Costs and availability are not the only considerations in the choice of a fermentation feedstock. There are other factors that need to be looked upon seriously
1 Agricultural or food industry wastes are often not of homogenous composition and its composition varies all the time
2 It is most likely that these sources of feedstock are not only contaminated by chemical, physical but also microbial contaminants
3 Nutrient quality or composition might be far from complete, ie not nutritionally balanced
4 They usually need to be pretreat before they are ready to be used as fermentation feedstock
5 They might cause complications during the actual fermentation process or downstream processing activities
6 Their product yield volume and quality might be low
PROBLEM AREAS IN FERMENTATION
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When choosing a particular fermentation feedstock, the two main problem areas created by the fermentation feedstock are in:
1 In the fermentor where the microbiological and biochemical transformations of the feedstock to product occurs
2 Down stream processing where separation, concentration and purification of the fermentation products from the fermentation broth occurs
Using a poor choice of fermentation feedstock will result in possibility of microbial contamination, foaming and mass transfer problems which will complicate the process and add costs to the production
I downstream processing extra efforts at additional costs are needed to extract and purify the products
IS IT WORTH IT?
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Some fermentation technologist even recommended using lab grade chemicals to be used as fermentation substrate to avoid complications in the fermentation process and increased cost in down stream processing. Of course on a large volume low value fermentation this is not really
posible......!!
Read more!
Monday, January 28, 2008
INTRODUCTION TO SCALE-DOWN AND SCALE UP IN FERMENTATION TECHNOLOGY
Two of the most common phrases one often met in fermentation technology research is 'Scaling up' and 'Scaling down' studies. However, the phrase 'scaling up' is more commonly understood and practiced during the designing of industrial scale fermentors. Where as 'scaling down' studies are rarely heard that frequent. In reality many fermentation technologists are not aware that during most times of their work they are doing 'scale down studies' Maybe the phrase 'scale up' has more impact factor than 'scale down' studies.
Let us simplify the similarities and differences between these two phrases in fermentation technology. A good example is when we intend to start with a fermentation process with the ultimate objective of producing the fermentation products on the level of industrial scale. Products need to be produced at large volume so that the process is economically viable. This requires scaling up. We do scaling up studies to ensure that the fermentation process is technically and economically viable to be produced in the end at a large scale
SCALE UP STUDIES
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Scale up studies are studies carried out at the laboratory or even pilot plant scal fermentors to yield data that could be used to to extrapolate and bui;ld the large scale industrial fermentors with sufficient confidence it will function properly with all its behaviours anticipated. More important during scale up exercises you are trying to build industrial size fermentor capable or close of producing the fermentation products as efficient as those produced in small scale fermentors
INITIAL SCALE UP STUDIES
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Most scale up studies are usually carried at different phases involving different scales of fermentors.Preliminary work are carried out at the level of petri dishes and small scale laboratory fermentors to establish whether the process is:
1 Technically viable, meaning it is possible to produce such fermentation process and the products on the small scale. Additional parameters not provided by petri dishes studies and for more confidence are obtained by carrying further studies using submerged liquid fermentation using various sizes laboratory scale fermentors and even a pilot plant fermentor.
There are a few rules of the thumb followed when doing scale up studies such as:
1 Similarity in the geometry and configuration of fermentors used in scaling up
2 A minimum of three or four stages of increment in the scaling up of the volume of fermentation studies. Each jump in scale should be by a magnitude or power increase and not an increase of a few litres capacity. Slight increase in the working volume would not yield significant data for scale up operation
It must be appreciated as the size of fermentation increases during scale up various parameters measured might not show a predictable linear co relationships. Certain parameters changes. Some remained constant. Some parameters need to be modified and adjusted during scale up studies. The objective is to try to get the same fermentation efficiency as obtained in small scale fermentors at the most economical values
IF SCALE UP STUDIES FAILED?
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Should at this stage the fermentation process is technically not possible and is a failure than we have two options:
1 Either find the cause of the failure or back to the drawing board!
2 Abort the whole project with minimum economic loss to the investors
The investors and engineers need more confidence in predicting how the behaviour of the fermentation will occur at the level of industrial scale. If it fails it might mean millions of dollars lost in the exercise where failure was not seen. So scaling up will give the investors more confidence on the chances of success and against economic disasters
The exercise in scaling up involved a number of programmed research or steps that has to be established so as to predict the final behaviour of the final large scale production fermentor. Studies carried out during scale up include:
1 Inoculum development
2 Sterilization establishing the correct sterilization cycle at larger loads
3 Environmental parameters such as nutrient availability,ph, temperature,dissolved
oxygen,dissolved carbon dioxide,
4 Shear conditions, foam production
SCALE DOWN STUDIES
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In scale down studies the main objective is to carry out studies on smaller bioreactors in order to gain data and confidence and predict the behaviour how things actually will behave in large production fermentor. Scale down studies are also used while during the operation of large industrial scale fermentors in trouble shooting or trying to optimize the industrial scale fermentation. This method is called the fermentation monitoring experiment.
The goal when scaling down is to create a small-scale or lab-scale system that mimics the performance of its large-scale (pilot or manufacturing) counterpart, when both the process parameters are varied within their operating ranges and also when a process parameter deviates outside its operating range.
The main type of studies in scale down such as:
1 Medium design
2 Medium sterilization
3 Inoculation procedures
4 Number of generations
5 Mixing
6 Oxygen transfer rate
RULES OF SCALE DOWN
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In scale down a few rules that should be followed:
SIMILAR FERMENTOR GEOMETRY
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1 Similar model geometries and ratios of system. The impeller and sparger designs, and placements within the vessel must be identical or similar. Wrong models used in scale down studies might invalidate the data obtained. Since a typical fermentation process might involved different fermentor capacities,scaling down will therefore be very challenging and proper strategies need to be developed during scale down studies.
SIMILAR METHODS OF MEASURING ANALYZING SAMPLES
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Similar methods of analyses and monitoring be applied at scale down studies such as:
1 sample-dilution schemes and measurement times for calculating culture optical densities,
2 wet and dry cell-weights,
3 media metabolite levels.
SAMPLING VOLUME
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Due to the involvement of small scale fermentors which contained less working volume, the sampling volumes should be minimized to prevent depletion of culture broth beyond acceptable levels. If the sample size cannot be reduced, then adjust the frequency of sampling.
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Mass transfer of oxygen between big and small fermentors is a critical issue in scale down studies. The efficiency of oxygen transfer on production scale fermentor is much lower compared to the lab scale fermentor. The strategy in doing scale down studies in oxygen transfer is to maintain similarity in sparger design, calibration and placement within the small fermentor and the large fermentor. If the sparger design is different between scales, then agitation, aeration and oxygen enrichment may need to be adjusted to provide equivalent oxygen transfer in the small fermentor
INOCULA
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It is very important in doing inoculum development during scale down exercise to maintain the vessel geometries, incubation conditions, and working volumes whenever possible during the scale down exercise. If in the process it is not possible to obtain fermentors of similar geometries
the operational control parameters may need to be adjusted to account for different vessel geometries.
STERILIZATIONS
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During sterilization studies in scale down studies, the sterilization temperatures, procedures for probe calibration, and post-use cleaning protocols should be the similar as the large-scale fermentor.
FERMENTATION FEED
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The raw materials used in scale down studies should be identical to those used for the full-scale process.
FERMENTATION CONTROL PARAMETERS
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Similar operating regimes and controls should be applied to the small scale fermentor such as
1 process temperature
2 pH
3 inoculation percentages (v/v) for each step
4 schedule of feed-media additions.
A linear adjustment method should be used for all the volume-dependent operational control-parameter set points except agitation. The scale factor should be equivalent to the ratio of overall process volumes. Examples of linear adjustments in:
1 Pre-and post-sterilization volumes of growth media.
2 Feed media delivery rates.
3 Total airflow.
4 Oxygen flow rate.
AGITATION RATE
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Set agitation in scale down studies to provide either representative :
1 oxygen transfer rate
2 tip speed,
3Reynolds Number, or
4power-input per unit volume,
Under conditions of similar fermentor geometry it is recommended that the oxygen transfer rate studies be carried out.
CULTURE GROWTH
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Culture growth is a critical performance parameter for qualifying the scale-down studies.
Oxygen utilization is a very important performance parameter for scale-down exercise. Similar patterns in dissolved oxygen profiles, and oxygen and airflow rates represent comparability in oxygen usage by the cultures at each scale.
PRODUCT YIELD
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A biochemical finger print should be established for both large scale and scale down fermentation for comparison for similarity in efficacy
PROCESS SENSITIVITY
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Process-control sensitivity for dissolved oxygen, pH, temperature, agitation and feed delivery must be verified at the small-scale.
DO SCALE DOWN STUDIES A FEW TIMES
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In general, for greater confidence it is good toperform at least three small-scale runs to confirm reproducibility and to determine the inherent variability in the process.
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Sunday, January 27, 2008
ENRICHED OXYGEN FERMENTATION
Oxygen is one of the fundamental requirements in any aerobic fermentations. The oxygen used by the microorganisms used in the fermentation process comes from two main sources:
1 Oxygen incorporated in the organic substrate which are used directly in the metabolism of biosynthesis of new biomass
2 Oxygen from the air which is pumped through the fermentation broth and used by the microorganisms in their respiration activities as their terminal electron acceptors
In aerobic fermentation although we see the mandatory requirements for oxygen by the microorganisms yet it exhibits the characteristics of Dr Jekyll and Mr Hyde of metabolism. It is needed but if exceeded it can be toxic and can kill the aerobic microorganisms
Oxygen too face other limitations in the sense of:
1 Its limited partial composition in air
2 Its limited solubility in water
3 Other parameters such as solutes, and temperature which affect its solubility
4 That microorganisms can only use oxygen not directly from air but only in the form of dissolved oxygen
In most fermentation process using fermentors attempts to increase the supply of oxygen to the broth and microorganisms have always been in the form of:
1 Increasing stirrer speed or mixing
2 Increasing volume of flow
3 Increasing the air pressure
Using the above three parametric controls have unavoidably resulted in building very large and costly fermentors
One of the alternative methods of increasing oxygen supply lately is by the increase in partial pressure of oxygen or enriched oxygen in the fermentation process. There are reports of increasing the efficiency of the fermentation process using this method but at the same time negative reports are cited of increasing microbial toxicity and poisoning by enriched oxygen.
There is also increase in hazards using enriched oxygen that may cause accidental combustions
PROBLEM OF MICROBIAL OXYGEN TOXICITY
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Most accept that oxygen are toxic to anaerobic microorganisms. Exposure to oxygen could kill obligate anaerobic microorganisms. Anaerobes could not stand the toxic effect of oxygen because their physiology are not equipped with enzymes that can make the toxic oxygen harmless. Aerobic microorganisms on the other hand have these enzymes to neutralize the toxic oxygen molecules. But what most people do not know is that oxygen can be toxic to aerobic microorganisms depending on the situation of operation.
It has often been regarded that increasing the amount of oxygen to the microbes during aerobic fermentation is a good option to improve the yield of the fermentation. However, under certain situation of exposure time and concentration or pressure of oxygen the process might be detrimental. Try reading articles by JG Morris and JWT Wimpenny for better understanding of the oxygen physiology of aerobic and anaerobic microbes!
The microbes are killed by the oxygen because they are not adapted to the operating conditions of oxygenation. The only alternative is to adapt microorganisms to the high oxygen exposure before using them as inocula for the fermentation process
Read more!
OFF GAS FILTRATION
Generally in any aerobic fermentation process, we are faced with the situation of 'What comes in into the fermentor, must come out of the fermentor'. This is especially true in the case of the supply of air or oxygen into the fermentor. Usually large volumes of air are pumped into the fermentor usually at the rate pf 0.5 to 2 vvm. So in the overall fermentation run, the volumes of gas passing into the fermentor, through the fermentation broth and out through the exhaust gas outlet will be tremendous. At any instant a positive pressure will be maintained in the fermentor due to the air flow
The physical, chemical and microbiological characteristics of air entering and leaving the fermentor will be different and represent challenges in the fermentation industry.
The main problem faced in the introduction of air into the fermentor will be the prevention of microbial contaminants entering the fermentor that will create problems in the actual fermentation process. To this effect filters are often placed to prevent the entry of the microorganisms in the air flow into the fermentor. These inlet filters not only prevent the entrance of microorganisms but at the same time remove any other particulate solids or impurities in the air such as grease and water
At the other end of the air line will be the exhaust gas or called off gas leaving the fermentor through the condenser to the environment. In the past the nature of fermentations is such that putting a filter at the off gas outlet is not mandatory as the positive pressure sustained in the fermentor will prevent the entrance of microbial contaminants entering the fermentor from the outside environment. However in the redebt decades with the involvement of the fermentors in preparation of vaccines and cultivation of pathogenic microorganisms, there is now more danger of the actual microorganisms escaping from the fermentor to be hazardous to the environment.
In situation like this, sterilizations of the gas leaving the out gas outlet is mandatory. Sterilizations of the off gas could be achieved by sterilizations using heat or absolute filtration.
The filtration of the exhaust gas from the fermentor faced many problems created by the nature and characteristics of the off gas such as:
1 High moisture content of the off gas due through their passage through the fermentation broth
2 Large pressure drops as the gas leaves the off gas outlet
3 Higher amount of microbial particulate matters that got carried away in the off gas
4 Rapid drop in off gas temperature which leads to condensation of water and clogging of filters
This will lead to blockage of filters and increase in head pressure of the fermentor
Related links: AIR FILTERS IN FERMENTATION
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Saturday, January 26, 2008
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
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Friday, January 25, 2008
SOLID SUBSTRATE FERMENTATION- THE ALTERNATIVE FERMENTATION
(PICTURE TAKEN FROM ARS.USDA.GOV)
The popularity of the fermentation process has always been represented by the image of large stainless steel fermentors and the production of fermented based beverages such as beers and wines. Yet, unknown to most and not keenly looked upon by scientists is the fermentation technology of developing countries known as Solid substrate fermentation. Solid substrate fermentation technology has all these while been operating quietly as a rural technology and playing very important roles in the preparation of food for the masses. Food such as tempe derived by SSF of tempe represents important source of protein and vitamins to the masses. The SSF process by the fungi involved helped also in increasing the digestibility of the substrate.
Composting too is a SSF process! It is widely used in generating valuable fertilizers out of organic refuse.
FUTURE POTENTIAL OF SSF
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Although SSF has always been considered a traditional fermentation used more in food industries such as Tempe , cheese and koji fermentation, it has lately been seen as a potential fermentation technology to be exploited in modern fermentation industries of today.
Research are now carried out to create novel SSF that will be able to produce high volume of industrial enzymes and fermentation acids such as itaconic acids and citric acids and even for pharmaceuticals.
SIMILARITY OF SSF WITH LIQUID SUBSTRATE FERMENTATION
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In principle, there is no real differences between the popular liquid based fermentation and solid substrate fermentation. Both fermentation processes still involved the same components of the typical fermentation process such as:
1 Presence of the fermenting microorganisms
2 The raw fermentation substate to be acted upon by the microorganisms
3 The end products of the fermentations produced which includes biomass product
4 A simple concept of bioreactor for which the whole fermentation process occur
5 Provision by the bioreactor of the ideal environment for the fermentation to occur
The most significant difference shown by the solid substrate fermentation is:
1 The presence of the nutrients for the microorganisms in the form of a solid phase
2 The very minimal requirement for the liquid or water phase as compared in liquid fermentation such as CSTR
MICROORGANISMS IN SSF
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Due to the nature of solid substrate and minimal content of water, microorganisms that dominate SSF are the fungi. The fungi are adapted physiologically to live within low water activity compared to bacteria and they can tolerate high osmotic conditions such in solution of high sugar concentrations.
The mode of growth of these fungi by hyphal extension with active hyphal growing tip that search and exudes hydrolytic enzymes work well in SSF.
THE SOLID SUBSTRATE IN SSF
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In the case of the SSF, the solid substrate which constitute the food or source of nutrients for the microorganisms could be viewed as a solid but porous matrix.
This solid porous matrix can absorb and retain a minimum layer or film of water or liquid which support a relatively high level of water activity. The film of liquid surrounding the various solid porous matrix is where all the activities of supporting the activities of the microbes occur.
Effective mass transfer of nutrients, oxygen and waste products occur within this thin film of liquid. Due to the high concentration of fermentation products such as hydrolysed sugars occurring within this thin film of water. This layer of liquid is often viscous, thus explaining more the importance of water activity rather than the presence of free water in the metabolism and biochemistry of SSF. Under such situations where free water is often limited and relatively high water activity, it is not surprising that moulds, yeasts and bacteria will dominate.
The nature of the solid substrate matrix is important in the success of any SSF. It should be soft enough structurally, porous enough to allow higher surface area to volume ratio to maximize mass transfer activities of nutrients , oxygen and gases. The SSF should allow a degree of mixings so as to optimize the fermentation process.
KEY PARAMETERS IN SSF
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When we compare SSF with the normal liquid fermentation, there are four key issues which could be discussed.
1 Moisture content and water activity
2 Temperature and heat transfer
3 Ph control and contamination
4 Oxygen uptake
WATER CONTENT
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As previously discussed although SSF do not need to have high amount of water, the presence of water as capillary water around the solid particles is important. These capillary water are very tightly bound to the particles and not easily available to the microorganisms resulting in water activity that support mostly fungi.
The optimum Aw for growth of a limited number of fungi used in SSF processes was at least 0.96 whereas the minimum growth Aw was generally greater than 0.9.The optimum Aw values for sporulation by Trichoderma viride were lower than those for growth. Maintenance of the Aw at the growth optimum would allow production of fungal and avoid sporulation.
TEMPERATURE
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Fungal respiration is highly exothermic. Highheat generation by fungal activity within the solids lead to thermal gradients due to the limited heat transfer capacity of solid substrates. Heat removal is a crucial factor in large scale SSF processes. Conventional convection or conductive cooling devices are inadequate for dissipating metabolic heat due to the poor thermal conductivity of most solid substrates. This will result in non acceptable temperature gradients.
Sufficient heat elimination is carried out through the use of evaporative cooling devices. Aeration helps in the transportation of heat out of the SSF. It should be cautioned that excessive aeration may result in dessication of the process.
PH
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The pH of a culture may change in response to microbial metabolic activities such as the production of acidic metabolites. These acids will cause the pH to decrease.
AERATION
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The four main functions of aeration in SSFare:
(i) to maintain aerobic conditions,
(ii) for carbon dioxide desorption,
(iii) to regulate the substrate temperature and
(iv) to regulate the moisture level.
Solid state process allows free exposure of atmospheric oxygen to the substrate and rapid mass transfer of oxygen occur easily through the thin liquid film surrounding the particles.
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Wednesday, January 23, 2008
DILEMMA OF DOING LATERAL RESEARCH IN FERMENTATION TECHNOLOGY
It is sad to note that in a number of universities and colleges I have visited, the research projects that have been carried out under the banner of fermentation technology are eother:
1 Isolation and enumeration of strains for certain biochemical or pharmacological activity
2 Establishing shake flask studies or laboratory fermentation studies of the species isolated
These represents initial studies that are carried out in upstream activities. No efforts are done in the coming years to go deeper into the studies initiated by either going further scale up studies or attempts to commercialize the studies.
To make things worst, the so called preliminary research will forever remain preliminary with no intention to carry deeper studies on the fermentation process by carrying other engineering aspects of the research. The next year or so, similar studies will be carried on similar strain or species of microorganisms. The difference maybe now they are doing studies on strains or microorganisms isolated from other sources or niches
I have the gut feeling that these researchers are really not interested in doing further research in depth on the studies or they are 'bankrupt. of research ideas.
It doesn't make sense to me after decades of research in the fermentation process, the direction of research is only going laterally and not yielding concrete results that can be commercially exploited. It is just a time wasting research that will lead to no where but wasting time, ebergy and capital equipment.
There is no attempt to refine or further the research. No attempts to innovate or improve the fermentation research. Maybe it is just another ' Mickey Mouse' project????
The way I look at it before any real research is done in the fermentation technology, these researchers must identify the objective of the research. They should ask the following questions before doing the research such as:
1 Is this research looking into really new novel or genetically engineered microorganisms?
2 Will it yield new products that is never yet on the market?
3 If I do research with existing microbes will my research end up with a new improved and efficient process in terms of its productivity
If we cannot answer these basic questions then in terms of business sense there is no point in initiating the research as failure will be the only option and there will be no commercialization of the process
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Tuesday, January 22, 2008
WHEN ENGINEERS AND BIOLOGISTS COLLIDE!
It is common to see that in such a highly integrated field of biotechnology and industrial microbiology where there is suppose to be a fusion or input of knowledge between microbiologists and bioprocess engineers we have a situation where both parties do not really see 'eye to eye'. This has more than often resulted in process failure or the bioprocess do not really run as the targeted expections.
PENICILLIN FERMENTATION
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In the history of penicillin fermentation or any other antibiotics, the progress in design of fermentors have been slow or static. In fact there is not much change in the design of CSTRs until now. Nor has the engineering designs improvements yield drastic improvements in the penicillin yield! The increase in the yield or productivity of penicillin fermentation has been more to the improvement of the strain by geneticists, biochemists and microbiologists!!
WASTEWATER TREATMENT IN BIOREACTORS
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A very good example is seen in the design of bioreactors such as bioreactors used in biological wastewater treatments. Bioreactors in biological wastewater treatments are often considered the biggest man made bioreactor using mixed cultures and operating continuously under septic conditions.
It is really hard to see wastewater treatment plants working efficiently and most bioreactors ending up by not performing to the expectations
There are valuable lessons to be learnt from this situation. For decades it seems that the engineers have a field day designing, building and operating the various biological treatment plants. The engineers only see the process as a " simple engineering" process, whereby there is input of substrate into the bioreactor, and where the microorganisms are present easily working themselves out happily eating away the pollutants thus treating the wastewaters. They only describe the process by a few key parameters which they think and understand to operate and control the bioreactors. Usually the standard parameters are BOD, COD, SS among others,
They do not see there is the need to understand the various biological and biochemical processes occurring in the biological reactor. As far as they are concerned, they designed the bioreactors more as the structure and volume to hold a certain effective volume of wastewaters over a certain designed hydraulic retention time.
Sad to say in such designs they only use the so called standard calculations from standard engineering texts in civil engineering. They do not take into considerations the:
1 Type of microbes and biochemical reactions that will be taking place as function of time or space
2 The supply and utilization of oxygen by the microorganisms in the bioreactor
3 Adaptation and wash out rates of microorganisms
4 Effect of toxicity and cumulative effects of toxic conditions
5 Nutritional and physiological needs of the various microorganisms
6 Behaviour of the populations of microorganisms in the bioreactor
The way they act as if nothing bad will ever happened or that all the bad processes will be taken care by GOD or blame it on poor understanding and managing of the treatment plants by the plant technicians.
In a way, we cannot blame this situation from happening as what is important is that the factory or the plant must have a biological treatment plant to get a certificate of fitness or license to operate. Whether it work or not is secondary and not the problem to the engineers that build the treatment plant
Why do most treatment plants fail after some period of operation? In the first place we see the whole scenario of the plant failure as a chain reaction of errors starting from poor engineering design and operation which will ultimately affect the performance of the microorganisms leading to poor treatment of the wastewaters. It should be noted that with just any process the range of optimal operating conditions in wastewaters operate within a very narrow babd of operating conditions. Change in any operating parameter will either lead to changes in the activity of the microorganisms , or worst still the change will affect the change of many other parameters either simulataneously or linearlr in a series of related events.
Changes to the process can either be immediate such as ' poisonings' which will kill the microorganisms or the failure of blowers leading to immediate anaerobic conditions that will result in abrupt plant failure. The second change occur slowly and cumulatively over a long period which will lead to gradual process failure
It is strange that if we surf the internnet and look at the various suppliers and contractors that sell or build the various biological treatment all GUARANTEE that their biological treatment plants will work wonderfully! Just you wait and you will see if such claims are true. As Tsun Zu the millitary strategist said once " In war, you win by deception". Business is war! They will promise you everything until you installed the tretment plant.
It is amazing in a few sites of suppliers and contractors of aerobic biological wastewater treatment plants they will tell certain parameters such as BODm COD and SS but failed to indicate the Dissolved oxygen readings of their treatment plants!!!!! Mind you we are talking about processes that uses oxygen.
They dont bother coming clean. All they bother telling you is their magnificient track record of having built this and that at here and there. Rarely will they give you the data of the actual plant or lab performance feaibility studies! What you will get are promises and 'guarantees' that it will work!
I cannot see why they dont give the other additional data parameters as the wastewaters go through the various unit processes so that the customer can assess the data themselves or employ an INDEPENDENT CONSULTANT to vet the technical data? This would have ensured the buyer that they are not cheated and will be getting good value for their money and freedom from future headaches?
Maybe the engineers 'hate' the microbiologists for their ability to see the process. Or is it because they think they are righter than God? or they are always correct?
If this is so why do most....MOST..... MOST treatment plants failed?
The point is while it is true engineers are trained to design and build these bioreactors, they must really try to understand the processes that is occurring in the bioreactor to come up with better design. It is time they stop the bad habit of designing treatment plants based on the number of PE for loadings pf the treatment plant or reduction of BOD and COD based on some standard references
Major designs like this must involve feasibility and technical studies to ensure that the treatment plant really will work and their range of test loads
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FERMENTOR AS A TEACHING AID IN BIOLOGY
In this era of modern biotechnology and scientific wonders and discovery, it is high time that those involved in teaching biology to the students or in designing the biological curricula for secondary schools to restock and start reviewing how the subject of biology should be repackaged.
For so many years the teaching of biology in schools and even in universities have tried to resist the 'winds of change' that is sweeping globally on how modern biology or biotechnology should be taught. Maybe in the past during decades of stagnation the importance of teaching biology is still bent on the fundamentals of taxonomy and classification and structure and function. The basic syllabus still do not change. In fact there is no significant improvement in the teaching of biology between university courses and biology taught at the university level.
We cannot still stick on with the rigidity of the old biology syllabus on one hand and yet demanded the graduates of these schools to acquire the knowledge of modern biotechnology without making the necessary changes and paradigm shift in how modern biology should be taught!
We are not advocating that we chuck or throw off mundane biology into the trash bin! We are only asking that the syllabus be changed, reduced according to the present importance and new emphasis be given to the subjects of modern biotechnology in schools! The old traditional biology need overhauling with new approaches in the teaching biology by exploiting the new tools of modern biotechnolofy such as using fermentors in explaining the various biological principles and experiments
THE FERMENTOR AS A TEACHING TOOL
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I have downloaded the following for attention National Centre of Biotechnology Education of their efforts to promote the teaching and education of biotechnology especially in the use of fermentors in classroom
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A range of fermenters designed for schools has been available in the United Kingdom over the last 15 years. These have varied considerably in price, complexity and ease-of-use. | ||||||||||||||||||||||||||||||||||||||
In response to numerous requests from teachers and our own concerns about classroom practice (particularly with regard to safety) we decided, in 1990, to design a cheap and simple fermenter or bioreactor for use in the school laboratory and indeed, in our own lab. Much to our surprise, more than 1,000 NCBE Bioreactors have been sold over the last five years. We never imagined that this basic 'kit of parts' would prove so popular (we had always thought that teachers would look at our design, then develop their own based on it). | ||||||||||||||||||||||||||||||||||||||
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Details of this SGM-NCBE practical guide can be found in the | |||||||||||||
Points to consider before buying any fermenter system for use in schools include: | |||||||||||||
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Safety guidelines for practical work with microbes in schools | |||||||||||||
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DESCRIPTION | ||
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The NCBE Bioreactor consists of a borosilicate 500 mL conical flask with a wide neck. The flask is plugged with a large, autoclavable, silicone rubber bung through which several holes have been drilled. Through these holes, the following items pass: | ||
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Both the air inlet and outlet have 'Hepavent' air filters to prevent microbes from entering or leaving the vessel. One or two other ports are provided in the bung to take, for example, a pH electrode or thermometer. The assembled Bioreactor is autoclavable (although you need a tall autoclave to accommodate it). You will also need an aquarium air pump to aerate and mix the Bioreactor's contents, and a thermostatically-controlled water bath to regulate the temperature. | ||
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DOCUMENTATION | ||
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The Bioreactor comes with a 36-page, illustrated instruction booklet. This booklet is beginning to show its age. However, a major revision is currently underway, and the development of an accompanying practical package has recently been completed (see 'Practical fermentation' in the Publications section). | ||
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NCBE BIOREACTOR (SELF ASSEMBLY) | ||
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NCBE Bioreactor ..... £110.00 (GBP) | ||
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REPLACEMENT ITEMS | ||
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The following replacement parts are available for the NCBE Bioreactor:
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Please note: All the prices on this page are in GBP and do not include Value Added Tax (VAT). This tax applies within the European Union only. Postage and packing must also be paid on orders from outside the United Kingdom. Please note: We do not currently accept orders via eMail. | ||
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Copyright © National Centre for Biotechnology Education, 2007 | www.ncbe.reading.ac.uk | ||
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The extent you can use the laboratory fermentor for teachings depends on the limits of your own imagination and creativity. We can carry out simple experiments such as:
1 Growth curve studies
2 Enrichment of cultures
3 Biochemical inhibitor studies
4 Physiological groups of microorganisms
5 Microbial ecoloogy
6 Nutritional studies and many more Read more!