RHEOLOGY PART 5: LESSONS FROM A DROP OF HONEY!

Sometimes we can learn more about the problems of rheology and mixing of fermentation broth by observing simple examples such as a drop of honey. A drop of honey as a rheological model is not truly reflective of the behavior of the fermentation broth but its behavior will allow you insights of mixing non Newtonian fluid.
If we try to stir the drop of honey on a surface using a tooth pick, we will see that it is very difficult to mix the drop of honey homogenously. There is movements by the tooth pick, but in most cases the honey will try to resist the movement and retract elastically back to its mass. Even if mixing occurs temporarily, it only occurs within the close proximity of the stirrer. Increasing the speed of mixing at most times does not increase the mixing of the honey.
Are we trying to say that in fermentors with very viscous broth mixing comes to nothing? Or better still have we come with properly designed stirrers that can really effectively stir the fermentation broth?
The biochemical engineers need to understand more about the properties of the non Newtonian broth and designed new stirrer configurations or even new modes of mixings to overcome this problem. Maybe it is high time or over time that they should start to look at the micromixing aspects rather than be over whelmed by macro mixing properties




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MICROBIAL GROWTH CURVE PART TWO :STATIONARY PHASE- FACTS AND FALLACIES

In the study of fermentation technology, we rely strongly on the understanding and interpretation of the microbial growth curve in making the right decisions for different stages of the fermentation process. We use the growth curve not only to determine the efficiency of the fermentation process but also in trouble shooting exercises
Yet, despite its importance how much do we really understand about the microbial growth curve? In fact, most of us learn about the typical sigmoid growth curve even as early as out initial years in our secondary education and right up to our university days. But really how much do we know about the microbial curve?
Instead we have been indoctrinated by superficial ‘brain washings’ of knowing the lag, log and stationary phase and the verbal description or verbal description of the phases of growth; slow growth, exponential growth and no growth…
They keep telling us the stationary phase is the onset period before the microorganisms die. They tell us that the stationary phase occurs because of lacked of food or toxic conditions. And that the stationary phase is a useless phase of no importance to physiology!
THE TRUTH IS FAR FROM THAT!!!
In terms of microbial physiology, the stationary phase is the most important phase when the microbes will undergo huge changes in its metabolism (prior to kicking the bucket!) It is the time for some microbes to produce secondary metabolites such as the antibiotics or even form spores to survive.
Before going into further detail let us try to understand what factors cause the induction of the stationary phase. Bear in mind we are dealing not at the behavior of single cells but a population of millions and millions of cells of diverse types of metabolism and physiology
The stationary phase the growth curve is often visualized as a level phase occurring after the end of the log phase and before the start of the declining or the death phase. Since the growth curve is a graphical representation of mathematical data, we could therefore say that at the stationary phase, the numbers of cells remain constant where either there is no growth or loss of the population of cells or more correctly where the number of new cells added is the same as the number of cells lost. It is hard to imagine that at the stationary phase the cells are not growing at all! It is more of a steady state where the number of cells remained constant
One of the most common explanations to explain the formation of the stationary phase is when substrate becomes the limiting factor. It is a situation where there are not enough nutrients to support the growth and multiplication of all the cells.
Another hypothesis is that stationary phase is induced by the presence of toxic products which reach a certain concentration to inhibit the growth of the microorganisms.
What is often not discussed is the impact of viscosity that might have impact directly or indirectly on the induction of the stationary phase. Then it is quite fair to say that limiting nutrients or substrate are not the only factor initiating the onset of stationary phase.
With regards to toxic products build up inducing stationary phase it might be quite valid in cases where there occurs catabolite repression or the fermentation products itselves are too toxic or too acidic. This argument however does not hold where in aerobic metabolism the end products are just water or CO2
Now let us look closely at the problem. The formation of the stationary phase is commonly associated with the sigmoid growth curve of try phase is commonly associated with the sigmoid growth curve of the microorganisms in general. It is more applied in batch fed mode. Why is this so?
It is more of the continuous washout rate occurring in the fermentor where the growth of the microorganisms is controlled by controlled wasting of cells, raw substrate and waste products. You can initiate continuous phase at various point of the log phase….
I tend too see the onset of stationary phase as a major shift in microbial metabolism rather than the onset of the equivalent of menopause….:) It is a point of intense change in the metabolism of the cell when the microbes are facing extreme stress with regard to their survival. The microorganisms have to carry out drastic changes in structure and function to survive or to insulate the integrity of their metabolism from the normal mode until better conditions will return
However it is during these trying times that the microorganisms will conjure up new compounds which do not seem to serve any functions to them. Initially it seems that these moves are stupid as the microorganisms seemed to waste energy, enzymes and carbon at time when they should be trying to conserve these precious supplies.
Could it be that evolution that took billions of years is stupid or more better we are ignorant of the strategy taken by the microbes? Sometimes we have to change our paradigm and try to see the significance of things from the point of view of the microbes rather than trying to do the thinking for them






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PROBLEM OF MYCELIAL SUBMERGED FERMENTATION

Get a large beaker of water. Fill it various strands of fine cotton strings of varying lengths. Then slowly switch on your stirrer. Observe what will happen?
You will observe that as the water starts flowing and mixing, the strings will start getting entangled not only with other strings to form pellets, but also with the spinning shaft and impeller.
This is what will happen too during submerged mycelia fermentation involving fungal or streptomycetes with hyphal structure. Strands of hypha will behave like the strings in forming pellets or getting entangled with the impeller or shaft. The hydrodynamic characteristics of the broth during mixing will cause collision between the various hypha resulting in the formation of pellets.
Of course the use of the string is just a simple model to explain what happens in the broth turbulence. In the real situation involving the mycelia the effect of mixing upon submerged fermentation is far more complex
A lot of submerged fermentation is involved in antibiotic fermentation. Despite the decades of experiences in mycelia antibiotic fermentation, there is relatively poor understanding of the behavior and physiology of mycelia in fermentation which resulted in poor control of the fermention process.The behavior and growth forms of the mycelia in the fermentor affects various mass transfer processes and even the microorganism itself.
The fermentation of the antibiotics initially requires the build up of large amount of biomass in the trophophase before secondary metabolism could be initiated to form antibiotics at the idiophase. Large amount of mycelia will be generated in the log phase prior to the onset of the stationary phase.
Intense aeration and mixing at this stage will result in collisions of the hyphae and mycelia forming fungal mats, pellets and other microbial aggregates. High shear forces are generated at the impeller tips, blades, and fluid turbulence and even at the bubbles paths.
On one hand, such shearing forces will damage the cells or hyphal and thus affecting metabolism and antibiotic formation. While on the other hand such hydrodynamics will form microbial aggregates which will affect the mass transfers across the aggregate and even the viscosity of the broth
The fungal pellets in antibiotic fermentation are interesting in their own way. The morphological and biochemical characteristics of the fungal pellets change as the fermentation progresses
However we can divide the fungal pellets into three classes:
1 Open pellets
2 Semi dense pellets
3 Dense pellets
The open pellets are not dense showing diffuse growth of hyphae,fused together and autolysed while the dense pellets are darker inner mass with thicker hyphae and healthy apical tips. Fungal pellets are larger compared to streptomycete which are smaller and simpler.
Correct shape and form of pellets indicate fermentation going as expected. This is useful diagnostic tool to indicate the onset of secondary metabolite formation during the antibiotic formation




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RHEOLOGY PART FOUR: APPLYING RHEOLOGY TO FERMENTATION

In most, if not all of fermentation broth, we are dealing with Non Newtonian fluid. The Non Newtonian nature is due to the composition of a fermentation broth which is not uniform and complex. The fermentation broth often show complex interactions of solid, liquid and gas phases.
To make things worst the rheology of the fermentation broth is always changing as a function of time and with the progress of the fermentation process.
It is more difficult to control and optimize a fermentation process if it is a Non Newtonian fluid! Things would definitely be easier if the fermentation broth is a Newtonian fluid. (But then again there would be no bread, cheese, yogurt, fish sauce and many more fermentation products!)
The main impact of Non Newtonian rheology is that it affect mixings and mass transfers of heat and oxygen and prevent efficient homogenous composition to occur.
We all know that in rheology it is the study of fluid deformation and flow under pressure and the relationship between stress and strain. Through simple observations we can see how difficult it is to mix and aerate viscous fluid. Each rheological type will give different mixing profile.
This has led to the classification of various classes of Non Newtonian fluids such as
1-viscoplastic fluid,
2-bingham fluid,
3-pseudoplastic fluid,
4-dilatant fluid
Non Newtonian rheology curves can be made up of various types. Most of these rhelogical curves are graphs where the x- axis is shear stress and the y- axis is shear rate
The rheological graphs are interesting not only in comparing between the Newtonian and the Non Newtonian but also the varying properties even among the various Non Newtonian fluid
It is interesting to note generally that all Non Newtonian fluids show some similarity in relationship with Newtonian fluid reflecting the effect of shear stress on shear rate. There is roughly a direct linear relationship (with variations) between shear stress and shear rate.
Differences only that Newtonian fluid adhere strictly and very linear and start at point zero of the axis.
1 Viscoplastic and Bingham starts only after certain level of shear stress. This means that the fluid will NOT respond immediately to applied stress and will only react after reaching the critical power point
2 Pseudoplastic and dilatants start at zero point but are curved in their shape. This mean that the fluid will respond immediately to the power or energy input. This is similar to Non Newtonian. However their response will be different in that it is not a linear relationship between stress and shear rate
3 Viscoplastic, pseudoplastic and dilatants are curved in their shapes This means that these fluids react in their yield behavior under stress differently.
So what does these observations mean in fermentation?
These rheological graphs will tell us how to respond efficiently with the type of broth being fermented.By understanding the various rheological changes that occur in the fermentation broth we can:
1 Try to achieve uniform homogenization and optimum mass transfer
2 Try to optimize energy usage in mixing of the fermentation broth
In carrying out the fermentation, we are using the impeller to mix the broth. Energy is transferred and dissipated to the broth by the impeller system. The impeller is in simplicity the shear stress being enforced upon the broth.
The effect of the impeller or mixing on the broth will result in the flow or turbulence of the broth. The broth will respond by exhibiting stress yield properties such as thinning out of the broth to improve mass transfer processes.
So if we know the rheology of the fermentation broth it will help us to adapt to obtain very efficient fermentation by adjusting our mixing regimes. This is especially so when the rheology changes with time and conditions.




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FERMENTATION METABOLIC PATHWAYS

We have always looked at fermentation process or the formation of fermentation products by microorganisms as something beneficial to man. While we are deriving the benefits from the activities of the fermentation microorganisms, in reality the action of these microorganisms are really not to serve the human masters but more to their selfish ends. To these microorganisms, the fermentation activities are their mode of survival. Fermentation is their metabolic and physiological method of deriving energy from the substrates for their growth.
The fermentation products during their metabolism are just their metabolic waste products which have served their duty. If however, the human beings get their pleasure by consuming their waste products……well and good for these pathetic humans. Nothing is stopping these humans from drinking their metabolic urine or alcohol !
In fact the cornerstone of human civilization in history has always depend on these fermentation products. There is a rich diversity of fermentat ion products as reflected in the culture of various countries. In essence, the diversity is quite limited in terms of the microorganisms and metabolism involved. The fermentation products are just given different brand names in different languages and by the use of different substrates found in the specific countries
The fermentative microorganisms metabolize the substrate to extract their enegy. This is executed by various metabolic pathways found in the microorganisms. It should be stressed that there is a diversity of metabolic pathways available, depending on the type of microorganisms, substrates and physiological environment. What is important to note are:
1 There are a number of fermentation pathways available in a microorganism
2 TCA cycle is only available in aerobic microorganism, BUT at the same time these aerobic microorganisms too have anaerobic or fermentation pathways . These microorganisms too are carrying fermentation process in their metabolism
Most students, in their early stages of learning microbial metabolism erroneously tend to think that only one or two pathways are involved. Or they only tend to think that in the study of fermentation metabolism only carbohydrate catabolism is important! Nothing could be further than truth in these assumptions. It is like saying lipids and proteins are not used as substrates in fermentation.
The beauty about having many fermentation pathways interlinking allows the fermentation microorganisms adaptability in the uncertain environment....and their survival!!!
The changing of tracks in metabolic pathways are achieved by the presence of branch end compounds. Branch compounds are compounds which interlinked two or more metabolic pathways. On reaching a branch end compound the microorganism will have the choice to go for one or other pathway depending on the circumstances. A very central branch end compound is pyruvate. From pyruvate the metabolism can go almost any where.
Such branch end compound such as pyruvate allows the effective linking of carbohydrate, lipid and protein metabolism. It also linked the various anabolic, catabolic and intermediary metabolism.


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BIONEXUS: COMPANY PROFITABILITY OR NATIONAL INTEREST?

Everyone knows the greatest power in driving any company is profitability. To increase the company profits and to reduce its risks companies are known to cut corners and finding ways to reduce the taxes they have to pay. Even better if there are other parties who are willing to inject capital especially in their high risk projects! Things simply could not go wrong! It is the ultimate formulae for success at least for the companies
Every body knows that setting up companies involve a lot of risks and it is a well known principle that out of the many new companies formed, many will fail. This failure is attributed to many reasons ranging from poor management, technical problems, and marketing and even from oversight to cash flow problems.
Some projects are even doomed to failure even before taking off, or while still on paper. They just don’t learn!!!. The best advice you can get before starting a company is to try obtaining the fundings from the bank or any other private institutions. They will vet your paper to the dot to see if the project is going to be a liability to their investment
And suddenly in the exciting spin of biotechnology, we have people or bodies who are willing to throw caution to the wind and throw “free or easy” capital to these companies in the name of biotechnology! The only problem is the money they are throwing are not theirs but the taxpayers!
As for me I would not be convinced by hype sounding words or terms, nor promises or potentials! To me I want to see track records. I know there are risks involved in any project but at least I will take calculated risks! I am not convinced at all by the sacks of MOUs but rather by the company performance and profitability.
I fear that in the end all these bionexus exercise will only profit the companies and not the nation


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LAMINAR AIR FLOWS IN FERMENTATION ACTIVITIES

It is very difficult to imagine running a fermentation plant without a good air supply. Air in various forms and quality are needed to carry out the various activities in the fermentation right from upstream to downstream activities.
Today, we will be discussing about the use of laminar air flows in fermentation activities. Laminar air flow is the term we use to describe the flow of air which occur in parallel layers or sheets just like a laminated sheet in cross section. The easiest way to visualize occurs air flow is to observe the laminar flow in liquid during primary mixing.
Conditions of generating laminar air flows occur under low energy power. High power usage will generate turbulence. Laminar air flow may further be enhanced by the type of nozzle used and by using laminar devices.
Laminar air flow are used more in the belief that such air flow do not create turbulence that might result in increased loading of microparticles in the air.
What is often not appreciated there is turbulence associated with laminar air flows by mechanically disturbing the immediate environment. Second turbulence is created as the laminar flows get mixed and merge. Points of energy cancellations will result in fast sedimentation particles
Laminar air flows do effectively transport microparticles at the zone separating the various layers of air.
The use of laminar flow is usually only effective in small space confinement such as in the inoculation chambers where the positive pressure exerted within the chamber tend to repel any intrusion of particles from the outside.
There is the trend nowadays to incorporate gentle laminar flow on the top surface of the fermentation floor to sweep away the microparticles.
It is regretful despite the understanding of the properties of laminar air flow its incorporation in the architectural design of aseptic areas in sensitive fermentation areas or even in the design of surgical operating theatres are still poorly understood

It is therefore not surprising in surgical operating theatres higher infections are often associated with the use of laminar airflow?


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RHEOLOGY PART THREE: DISCUSSIONS

One of my students used to ask me this question,
“What is the point of trying to understand the complexities of fermentation rheology, when rheology itself is not one of the common parameters used in monitoring the fermentation process?”
I threw this question to the class for deeper discussions..
The general consensus is that:
1 Rheology is an important and relevant parameter of understanding and controlling the fermentation process.
2The use of rheology part could contribute significantly in understanding the changes that occur during the fermentation process.
3 Rheological data could be used in trying to optimize the conditions towards optimum fermentation process.
4 Rheological data could be used to extrapolate the likely event of impending fermentation failure
5 The use of rheology data such as the type of non Newtonian fluid could indicate the right opportunity to change the mixing regime and save energy
6 Operating parameters could be adapted such as mode of feeding to control the rheology of the fermentation broth
7 The failure to use rheology as the online parameter is because there are no sensors that can measure rheology on lines for now, and not because rheology is not an important parameter!


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PROBLEMS OF VOLATILE FERMENTATION PRODUCTS

In the true tradition and history of fermentation, the reference to fermentation products has always been associated to fermented drinks and foods. These fermentation products are products excreted by various fermentative microorganisms in the absence of oxygen.
These diverse fermentation products have always been characterized by their unique chemical and physical characteristics which give them their own unique taste and odours.
The fermentation products are simple organic compounds usually with carbon skeleton between one and three carbon. The short chain compounds are characterized by their volatility at room or ambient temperature.
The aromatic nature of these fermented products are attributed to their own unique chemical groups such as volatile fatty acids, ketones, aldehydes and even the presence of sulphur containing compounds. It is the interactions of these various volatile which give the unique odour and taste to butter, fish sauce and wine among others.
The problem is that in the age of industrial fermentations there is the need to produce large volumes of these compounds. Some of these fermentation products need to undergo various processing treatments to make the product safe and acceptable. Some of these products need to be modified and repackaged to fulfill the needs of the modern consumers.
What are the effects of these processings and treatments to the quality of the fermentation products? This would not be a problem if the fermentation products are simple compounds produced by single cultures and using modern fermentation technology such as beers. This would however be a problem when the fermentation products are a mixture of volatile organic compounds produced by mixed culture fermentation such as in fish sauce or budu fermentation
Definitely such processing treatments using high temperature will affect the composition and quality of fermented products. The powdered fermentation products when reconstituted will definitely taste different or lost that characteristic bouquet!
Maybe it is for that reason certain fermentation products will always remain traditional…..and tasted better





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