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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WEISENBERG EFFECT- PROBLEMS OF STIRRING BIOPOLYMERS IN FERMENTORS

It is the purpose in any engineering design of a fermenter to achieve uniform and homogenous mixing. This objective is to ensire that the content of the fermenter is homogenous and good mass transfers occur between the microorganisms and the surrounding environment.
Frequently though this objective is always kept in mind, the weakest link in the process is the failure to understand the rheological properties of each fermentation broth and the provision of unsuitable stirrers or mixers to achieve this end.
Complications arise due to the fact that most if not all fermentation broth are non newtonian and show very complex properties spatially or even temporally during the fermentation run. Just providing any standard stirrer is not the proper solution to this problem. Each fermentation process have its own unique problems and conditions and almost require its own specific stirrer and mixing regime.
One good example often faced in fermentation industries is the problems of mixing viscous broth. This is often encountered in food fermentation such as yogurt fermentation or fermentation of biopolymers or those involving use of sticky sugar substrates.
In theory we expect any mixing of fermentation broth would result in homogenous conditions, improved mass transfers. In viscous broth we expect the effect of stirring would be to stretch and thin out the broth to improve mass transfers
The problem is during the fermentation mixing or stirring there is the tendency for the broth to creep up the stirrer shaft, instead of being dispersed throughout the fermenter. This effect is called the Weisenberg Effect
The Weisenberg effect refers to a common phenomenon when a spinning rod is placed in a solution containg liquid polymers. This will result in the entanglement of liquid polymers to the spinning rod or shaft leaving the free ends of the polymers in the solution. Tensional forces acting on both ends of the biopolymers will try to reduce the distance between the two ends resulting in the polymers to move along the shaft.
Given time a mass of biopolymers will be cumulated at the end of the spinning shaft of the stirrer.
IMPACT OF WEISSENBERG
1 Extra load at tip of moving shaft will generate extra torque and if the shaft of stirrer is long will throw out the shaft out of its normal oscilation
1 Inefficient mixing as the impellers will be covered by the cumulated biopolymers
3 Entrapment of biomass that will not contribute to fermentation product
4 more downstream problems and loss of time in cleaning the stirrer system

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MAKING YOGURT- PLAYING WITH VISCOSITY

Almost everyone knows how to make yogurt. Yogurt is after all a simple fermentation process using milk as the substrate. The microorganisms involved in the process are . S.Thermophilus, L.Actobacillus Acidophilus and B. Bifidum.
In the process the milk sugar or lactose is converted into lactic acid and other fermentation products such as carbon dioxide, acetic acid, diacetyl and acetaldehyde which contribute to yogurt's characteristic taste and aroma.
It does not take a food scientist to make a simple yogurt. A hobbyist who is willing to ‘experiment’ with ingredients and conditions, and after passing through trial and errors will ultimately be an expert in making his own unique yogurt. p(Though others might not like his product)
However, if you are trully interested in making the perfect or standard yogurt that is marketable you do need to really understand the processes ivolved in making high quality yogurt.
Of course, the preference for a particular type of yogurt depends on the individual. But you need to produce a yogurt product of the highest quality, consistency.
A good yogurt is characterised by its flavour, aroma, appearance and texture. The texture of the yogurt itself is important. Many yogurt are designed to help create or maintain a thick texture.
TEXTURE OF YOGURT
Most people would like to imagine yogurt as viscous but soft enough to flow.The texture of yogurt is measured in terms of its flowability or viscosity.
The manufacture of yogurt itself in reality is complex as the rheology of yogurt fermentation changes with time and stage of process . This would not be a critical issue for home made yogurt involving small volume production
In controlling the viscosity of the yogurt would involve various stages of manipulations such as:
1 The milk used for making yogurt must be standardized and should contain at least 3.3% fat and 12 - 18% Milk Solids
2 Homogenisation should be used to increae the product viscosity
3 Pasteurisation at 98 degrees Centigrade for 3 minutes will help in the coagulation
4 Filtration should be used to removed pockets of whey in the broth
5 Cooling should be carried out in a controlled manner to maintain the integrity of the yogurt viscosity
6 Transfers of yogurt should be carefully carried out by pumping and mixing properly to maintain its viscosity




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MILK FERMENTATION- PASTEURISATION AND STERILISATION

Milk represents one of the most common source of nutrients used widely in all countries and human cultures. There are many kinds of milk available depending on the type of animals used to produce the milk. However, dairy milk or cow milk represents the most popular type of milk used by man.
Milk is so nutritious that not only its ideal for human nutritional requirements but also for the calves for which nature intended it to be in the first place.
It is a sad and tragic fact that lactating cows are the ones used to produce milk which we need in volumous amounts. To do so, these cows are needed to be fertilized, and only after they give birth to calves would milk be produced. Since the cows are meant to be producing milk, I often wonder what happens to the young calves produced once the dairy cows start lactating. Will they be allowed to grow into maturity or will they be slaughtered young ending up as veals?
My apologies for the temporal distraction from the topic…. Just cannot help it!
BACTERIA LOVE MILK TOO!
Milk being nutritious will also proves to be irresistable to the bacteria. Milk is rich with proteins and various vitamins and growth factors is too good a deal to be rejected by the bacteria. Bacteria especially those that are nutritionally fastidious will grow well in milk.
This nutritional attraction for the bacteria will have serious implications for the humans that drink the milk. Not only will the humans have to compete for the milk with the bacteria but the bacteria will result in the spoilage of the milk itself by its biochemical activities. The bacteria itself will be a source of microbial pathogens and diseases.
CONSEQUENCE OF MILK MICROBIAL CONTAMINATION
One of the consequence of microbial contamination and spoilage of milk will be short shelf life of the milk.Attempts were made to solve the above problem not only due to health but economic consequences.
One of the most common ways to reduce microbial contamination of milk is either by killing or removing the microorganisms in the milk.
There are several methods to achieve this but each method will have its own advantages and limitations.
The use of heat or high temperature is commonly used in pasteurisation or sterilisation of milk.
PASTEURISATION
One of the earliest method used in industries to solve the problem is by the process of pasteuerisation. This process is named after the famous microbiologist Louis Pasteur.
In the pasteurisation process the milk is subjected to a high temperature for a period of time which will allow the killing of some of the microbes without really damaging the quality of the milk
In the Pasteurisation process the presence of microbes is reduced…. (REDUCED) and not completely sterilized! The effect of pasteurisation process will be making the milk safer and prolonged the shelf life of the milk. This is of important consequence to the milk industries as more milk can be distributed wider and more profit
It should be noted the concept of pasteurisation is relevant more in the past when refrigeration is not a common household item.
In pasteurisation we see the reduction of the microbes occurred as the consequence of heat being applied. The MAIN OBJECTIVE in pasteurisation is the reduction or destruction of the microbes in the milk.
Pasteurisation is never seen as what EFFECT the process has on the nature and composition of the milk itself and its nutritional value of the natural milk itself
Milk being dominantly protein and amino acids with vitamins and growth factors are affected by heat. Pateurisation will alter the values of the milk itself.
Heating to this temperature, but no higher, does not change the proteins in the milk or the yoghurt so the taste is not really affected.
MILK STERILISATION
The most popular alternative to milk pasteurisation now is using the ultra heat treatment. In UHT the milk is heated to at least 135°C for at least one second. UHT destroys all bacteria in the milk and makes it last much longer than ordinary pasteurised milk. How ever using UHT does cause changes to the taste of the treated milk due to the denaturing of some of the protein components in the milk.





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