ASEPTIC TRANSFERS IN STRINGENT FERMENTATION INDUSTRIES

Microbial contamination of fermentation processes could be serious and even critical in some fermentation industries. The presence of unwanted microbes could not only jeorpadise the safety and quality of the fermentation products as in the production of pharmaceuticals but also cause product spoilage and short shelf lives of the products with serious consequence in terms of economy and time wasted.
Strange it may seemed but quite a number of people sees the problem of contamination only at the level of mid stream fermentation and ignoring the input of microbial contamination at both levels of upstream and downstream activities. They are in the belief that once a fermentation product is formed microbial contamination is no longer a problem.
In reality, the seriousness of microbial contamination occurs at all levels and at all times. In fact it is even more critical if traces of microbial contaminations occur upstream as the microbial contaminants will have time to amplify over space and time. And you will find yourself dealing with a bigger problem of contamination if steps are not taken to minimize the contamination problems earlier.
Finished fermentation products are attractive as substrates to microbial contaminants downstream. To the microbial contaminants these finished fermentation products are good food sources to support their growth
Everytime microbial contaminations occur in the fermentation industries it becomes a nightmare for the operators. The consequence of such contaminations would often mean the rejection of the complete process in terms of fermentation products, fermentation media which have very serious economic consequence. Costly steps are taken not only to terminate the fermentation but in washing, cleaning and eliminating the source of contaminants. Labour, downtime loss is part of the price they have to pay!
This situation is even more critical in pharmaceutical fermentations.
One of the main sources of introducing microbial contaminants occur during the transfers from one stage of the fermentation process to the other. We will now discuss the problems of aseptic transfers or aseptic fillings of the finished fermentation products in detail
The most significant word which we must contend and understand clearly here is ASEPTIC. Aseptic describes the condition where the environment is free from the presence of microorganisms which might cause the contamination. In a way it almost describes a sterile environment or zone where the desired activity takes place.
This situation of being absolutely free of microorganisms is almost quite impossible to achieve, especially in the environment of the factory floor or the fermentation plant. At best we can control only a certain volume of space as being free from microorganisms where aseptic transfers can be quickly carried out
It is of importance to use a smaller room for aseptic transfers in terms if economy and efficiency
Surfaces in the aseptic room must always be clean sterilized or disinfected as a matter of routine procedure
Microbes being microbes are very ubiquitous and microscopic. They are not easily seen and are easily transferred by air, water and other medium. A slight turbulence will easily dislodge and transport them from one point to another. Thus sudden or lots of movements should be avoided in areas where aseptic transfer are being carried out.
This is especially important when supplying air into the aseptic filling room.
Additionally the air introduced into the aseptic filling room should be sterile and filtered using a suitable size filter that meet HEPA standard
Cutting the air movements and turbulence is an important step in preventing microbial contaminations. The use of laminar flow is very strongly recommended to avoid turbulence
One of the greatest source or reservoirs of microns are the workers themselves. The human body carries a lot of microbial flora especially on the skin and our respiratory system. A sick or infected worker will even harbor more unwanted microbes and a source of disease and infections
The more workers you have the more risks you will face with microbial contamination. Keeping the number of workers to a minimum during aseptic transfer is important in reducing microbial contamination
The use of clean sterile protective clothing and guard help in preventing transfers of microbial contaminants from workers to the products
If possible most of the aseptic transfers should be automated or semi automated to prevent contamination from the operator



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CONTRIBUTION OF FLUID MECHANICS TO FERMENTATION TECHNOLOGY

Fluid mechanics is accepted as the integral part of study in civil engineering. It is also an important component in the study of physics. The study of fluid mechanics, especially fluid dynamics will try to explain the behavior and properties of fluid in motion. While fluid mechanics is important in the design of civil structures such as dams and bridges, it is of similar importance too in the field of fermentation technology, especially in trying to understand the behavior of the fermentation broth in the fermentors. Knowing the fluid mechanics in fermentors not only allow us to design better fermentors but will also improve the efficiency of the fermentation process.
In fluid mechanics it is important for us to appreciate that water or liquid could show two different behaviours under different conditions applied. We could treat the behavior of water as particle as in Langarian theorem or as a moving packets or bodies of flowing water in restricted volume in accordance with Eularian concept. Both approaches have their strength and weaknesses.
How does fluid mechanics has to do with fermentors?
A simple observation will tell you that all fermentors are equipped with some stirring mechanism of some sort. It could be in the form of stirrers with impellers or it could even be in the form of simple gyrating movements or waves in the case of the disposable reactors or fermentors.
The objectives of these stirrings or mixings is to bring about uniform composition throughout the fermentor and to improve its various mass transfer processes. Poor mixings and mass transfers will always result in sub optimum fermentation process.
There are many attempts to improve the optimum fermentation process by improving the efficiency of it kLa. But the solution is not as simple as increasing the oxygen flow or input. Attempts to increase the oxygen flow will lead to negative secondary problems such as shearing of cells and increase in foaming!
The type of fluid dynamics generated within a fermentor could be very complex. It might not only involved primary, secondary and even turbulent flow within the fermentor. This situation is further compounded by the size and geometry of the fermentors involved and by the presence of barriers such as the baffles and other structures. In certain situation the interaction of these liquid flows within the fermentor could lead to cancellation or even strengthening of the flow pattern and shear forces.
As we have said earlier in the mixing of the fermentation broth there are also very complex mixing of various sold, gas and liquid phases which respond differently to the mixing process and will affect the efficiency of the fermentation process
So how do we try to solve the problem of fluid mechanics that occur in the fermentor? The most likely answer will be the use of computational fluid dynamics in helping design and even locate the areas that need further attention

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THE QUESTION OF SIZE AGAIN.....PART TWO

It does appear that the discussion about the size of fermentors is not as simple as it may seem. In fact it is still a very complex issue and the point of contention among many fermentation technologists. Even till today, arguments on the pros and cons of choosing the right size of fermentors is still not settled.
There are generally two schools of thoughts on these matters. The first group who are advocates of going for large size fermentors or the scale up party, and those that goes for small and miniaturized fermentors or the scale down fermentation technologists
In reality both groups have their pros and cons. Every group has their advantages and disadvantages. What is right depends on the situation or the nature of the fermentation problems.
Size of fermentors was not an issue in the early days of fermentation. The simple rule the size of the vessel dictates the volume to be fermented. But with the advent of industrial microbiology where economics dictates everything, size and other parameters as efficiency, energy input suddenly becomes critical.
We have a golden rule in economics called the economics of scale. Where increasing volume produced will result in lowering the cost price of production per unit product. This often explains why fermentation industries have huge fermentors especially those involved in high volume low value products.
This rule could not be similarly applied to low volume high value fermentation products where other factors such as limitations in down stream processing is the constraining factor and purity of product is stringent
Lately in the last few years there have been a trend towards miniaturization of bioreactors or fermentors. This involves the use of fermentors of less than 10 ml or using of microtitreplates
The use of these very small bioreactors offer the main advantage of using small volume of media and allowing multi variate experiments to be easily carried out simulataneously or in parallel configuration. This is almost akin to the advantages of using solid media on petri dishes during primary and secondary screening.
The problem in using these miniaturized bioreactors differ fro the use of petri dishes in that it uses liquid media and tries to mimic what really happened in a liquid fermentation process.
This is not easy as the key issues in any liquid fermentation is attempts to get homogenous mixing, mass transfers and monitoring of the various fermentation process parameters.
The behavior of fluid mixing in miniature fermentors differs greatly from those larger fermentors where mixings can be carried out effectively by various mixing techniques from stirring to even shaking the conical flasks. In microbioreactors due to the small size the mixing of the liquid broth is hard to achieve especially due to the physical interaction between the liquid and the walls of the bioreactors. The phenomenon of surface tension and capillary effect will be significant.
Any new techniques to measure or detect efficacy of mixing in microbioreactors do have to depend in parallel development in techniques such as computational fluid dynamics.
The use of micro fermentor will generate its own set of unique problems not faced significantly when using large fermentors. Small volume of liquid broth will have higher surface area to volume ratio which will affect processes such as evaporation, surface tension. This if not controlled or taken care off will introduce errors in data to be used especially during scale up exercises .It doesn’t matter even if you have come up with miniaturized sensors the problem of mass transfers will be severely affected
Due to poor mixing any samples obtained would be questionable to its representative function. Wrong sample means wrong data despite the use of the most sophisticated microanalysers.
In my own personal view the use of microfermentors are still in the research stage and are of very limited applications in fermentation technology as of now 




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PRIMER FOR UNDERSTANDING FERMENTATION TECHNOLOGY- THE ENZYMES

Even though all these while we have credited the microorganisms as the transformation agents in the formation of fermentation products from substrates, the REAL heroes are the enzymes themselves that cause the transformation to occur. These enzymes produced or being part of the microorganisms are responsible for the changes
In a simple fermentation carried out in vitro using enzymes obtained from living cells it is possible to cause the desired transformation. In fact this classical observation is the event that gave rise to the birth of enzymology and biochemistry.
As far as the microorganisms are concerned, they are just living sacs full of enzymes that are needed to carry out the various metabolic reactions needed for life. In fact we can envisioned the living cells or cytoplasm containing protein molecules which are just enzymes especially in the cytosol. Its more like a balloon filled with a suspension of enzymes
Therefore to study or understand fermentation technology we need to study the complex interactions that affect enzyme activities.
The complication that arises in comparing enzymes in fermentation technology and simple enzyme reactions in biochemistry is that most enzyme studies in biochemistry are involved with simple enzyme system ( minus the living cell) and they are using pure enzymes and substrates. This simplify a lot of things!
Whereas in the living cell we are involved have many enzymes which influence each other and require the series of enzymes to complete the transformation.
The product of one enzyme is the substrate of the next enzyme. This is further complicated by different kinetics of each enzymes and different control of enzyme activities such as catabolite repression and product inhibition.
A look at the standard metabolic pathway chart will show you the flow of substrates, and points of intermediate diversion far more complicated than the Pudu Raya traffic interchange or London traffic 
The traffic system of the enzymes are not that chaotic as there are rules of enzyme reactions which must be adhered.
Knowing these enzymes are necessary in order for us to appreciate the various fermentation kinetics and to understand fully the importance of such equations such as Michaelis Menten and Monod equation.
So do smile as you try to understand the enzymes. They form the foundation of fermentation technology!

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FERMENTATION KINETICS PART ONE: WHY DO WE NEED TO STUDY IT?

In fermentation technology, we stress in understanding the various process in fermentor and how various intrinsic and extrinsic factors influence the fermentation process. Fermentation technology being an industrial microbiology subject are geared in producing maximum amount of high economical fermentation products
But it is difficult to understand and control the fermentation process as it involves various components such as effect of substrates, products inhibition, conditions and complex microbial interactions
The fermentation process is not only complex but always In a state of flux. Process, We are therefore in a situation to always be adaptive and reactive to these changes so that through out the fermentation process we are always sustaining the conditions in a narrow window of optimal fermentation conditions.
In order to help us to do this we need to know fermentation kinetics. When we talk about fermentation kinetics we are talking about fermentation models. Kinetics and modellings are very useful to us as tools to make fermentation predictions and enhancing our experimental designs to be more focused to the specific problems such as the rate limiting steps or product inhibition
The study of fermentation kinetics help us by providing clear quantitative data for us to understand the process and improve the process accordingly. Peering into observation ports might be good advertising gimmick for fermentation technology but do not really help much in understanding the process or even to control and predict the fermentation outcome. Subjective observations will rarely help in producing optimum fermentation process and thus affect profitability studies and making decisions
Its numbers that count! Real data that can be processed and determine decisions
Thus the importance of the study of fermentation kinetics or models
The first step in the study of fermentation kinetics is to understand the various processes involved in the whole process. Such questions such as inputs and outputs, the metabolic pathways involved and type of products or side products formed. The various individual reactions involved and what factors control the metabolite levels
At the level of the fermentor we need to know the various mass transfers involved, flows and mixing characteristics
Then only after all the relevant data are obtained do we start formulating the models



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MICROBIAL GROWTH CURVE PART 3: THE LAG PHASE

In a typical sigmoid growth curve, the first phase is called the lag phase. It has often been accepted by most that the lag phase is the period where there is no net increase in the number of cells. It is the stage where the cells are adapting to the new environment and are busy trying to synthesise new array of enzymes needed.
How true are these ‘allegations’?
As we have said earlier, the microbial growth curve is the graphical representation of the microbial population and not of a single cell. We are talking about millons and millions of cells. If we assume this statement that it is a period of no increase in cell numbers and it is just a period of enzymes induction then it is difficult to accept the idea.
Don’t tell me in the millions of cells there are no cells reproducing?
Even in a drop of culture or microbial suspension, containing millions of cells, each of the cell has different status in terms of its mass transfers exposure. Each cell are in different physiological states from young nd active to old and dormant cells.
Perhaps it might be logical for synchronous cultures to have same starting point in growth or lag phase. Even then synchronocity just last few generations.
We do know however that the length of the lag period is connected to various conditions from short for adapted cultures to long for cultures in a new environment. However that does not mean being in lag phase does not result in non reproduction of new cells. Maybe only the rates might not be significant.


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MICROBIAL GROWTH CURVE PART ONE- A SIMPLE INTRODUCTION

How do we define microbial growth? To most people when we talk about growth we are always talking about the progression in the development of the organism as a function of time. The progression of growth of the organism or the individual is usually associated with increase in size or biomass of the organism.
The key point here is the INDIVIDUAL organism. However in dealing with microorganisms, most microbiologists tend to picture microbial growth from the view point of the increase in the total population of microorganisms and not the individual unicellular microorganism
The growth of the microbial population at any one time represents the steady state number of cells or growth parameters used as the index of growth. The steady state numbers represent the net number of cells where input of cells and death or loss of cells are taken into considerations
The growth of the cells over time is often conveniently represented in graphs.
These microbial growth curves therefore represent the growth of microbial population rather than the individual cell. So any information derived from studying the growth curve represent understanding the behavior of the population rather than the individual cell.
The behavior of the individual cell differs from the behavior of the population of cells. This must always be bear in mind all the time in interpreting growth curves.
It is one of the weakest link in understanding the behavior of the microbial population to regard it as a simple integration of the activities of the individual cell. This is microbial physiology and not plain mathematics! Everything is not averages or mean values!
It is a gross error or over simplification to regard the microbial growth curve is the popular sigmoid shaped growth curve. In fermentation technology the type of growth curve obtained are determined by many factors and operating regimes. Yet time and time again the error of interpreting the wrong growth curves are continually repeated.


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