INDUSTRIAL PRODUCTION OF ENZYMES: PART 1 ENZYMES IN FERMENTATION INDUSTRIES

The good thing about the use of enzymes in various industrial processes amenable to enzyme activity is that the reactions are simpler, faster, easier to control and you do not have to worry much about unwanted side products or unwanted products downstream.
However in various industrial fermentations, the use of enzymes are restricted to a few steps which more than often are prelude steps before the real microbial fermentation process. The restrictions in the use of enzymes for industrial processes are often restricted by the type and suitability of the enzymes needed for the various processes in the complete fermentation process.
In industrial fermentations, the use of enzymes is more in the hydrolysis of substrates prior to its fermentation. This stage is often called the liquefaction stage where large molecules are broken down to simpler monomers to be easily used by the fermenting microorganisms. Enzymes too are widely used in the clarification process of various fermentation juices
Using the enzymes therefore help in making the fermentation process more efficient both technically and economically.
THE NATURE OF ENZYMES
Enzymes are basically catalysts, whose main function is to speed up the specific reactions. In a way, the enzymes are no better than the inorganic catalysts used in many of the industrial chemical reactions.
The differences between enzymes and the inorganic catalysts are more in the sense organic enzymes carry out their reactions under a less demanding conditions. Their reactions are more under gentler physiological conditions found in the normal metabolism of the cells.
Organic enzymes are usually large protein structures which are easily denatured by conditions of extreme ph, temperature and salts and metals. The activity of these enzymes are not stable as they are easily denatured . Their active sites where the reactants interact are very sensitive to such conditions
Industrial catalysts operate under very extreme conditions of pressure, temperature, chemical toxicities which would not be suitable for the living microorganisms.
There is high demand today for enzymes to be used in various food and beverage industries among others. One of the biggest outlet for the production of enzymes is by the use of fermentation technology where the enzymes itself is the sought fermentation products.
Before discussing further the production of enzymes using fermentation technology let us acquaint ourselves with the type and location of enzymes in the microbial cells.
Many new students tend to fantasize the enzymes as equivalent to the magic elixir that can transform almost anything to anything. As we have said earlier the function of enzymes are nothing more than speeding up certain biochemical reactions. The living cell which contains cytoplasm within the membrane sac is nothing more than a complex mixture or soup of enzymes. Without enzymes it’s doubtful life will persist!
In reality the function of enzymes are very limited to carry out certain chemical reactions which involve breaking or making new bonds between the chemical groups. We classify enzymes by the nature of the chemical reactions they carry out!
We also classify enzymes by their location that is they intracellular or extracellular. Or we can classify enzymes whether they are constitutive or inductive, or whether they are soluble or attached
The nature of thee enzymes as stated above will determine the conditions for the production of the enzymes and its downstream extraction isolation and purification
MICROBIAL ENZYMES
Most industrially important enzymes produced by microorganisms are those which are induced and secreted out by the cells into the fermentation broth. It is therefore of great importance and cautions that only suitable microorganisms are used in the production of enzymes using fermentation technology. Common microorganisms used are Bacillus subtilis and Aspergillus oryzae
Much work and research are needed before these microbial enzymes can be used in large fermentors. What is most important is that at least the microorganism chosen must have the ability to produce the desired enzymes. The productivity of the microorganisms can further be enhanced by using various techniques of biotechnology such as gene manipulation
The enzymes produced by these microorganisms do not come in market ready forms! A lot of work needs to be done especially in the downstream activities.




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BIRD’S EYE VIEW OF FERMENTATION PROCESS

One of the first things the students taking fermentation technology will be the outline of the fermentation process flow. He will learn that the complete industrial fermentation process will be made up of three main stages:
1 Upstream activities
2 Midstream activities
3 Downstream activities
In any industrial fermentation process is seen as a pipeline of activities originating upstream with the fermentation products finally being obtained at the end of the pipe or at the downstream stage.
Thus in a simplification in the process flow we see masses being introduced into the process and as it flow down the pipe these masses are being transformed to the desired fermentation products by the fermentation microorganisms.
At each stage of the transformation optimal conditions are implemented not only to obtain the highest yield of the products but also in terms of energy and economic efficiency. This attempts can only be carried out if we assumed that the flow of substrates through the fermentation process will pass through various unit processes which could be identified and optimized.
In such situation, it is important that:
1 The flow of masses from the beginning stages to the final products will be smooth and not interrupted. To achieve this hydrodynamic flow restrictions imposed by various volumetric and hydrodynamic restrictions must be overcome by using different bioreactor or volumetric configurations
2 That each unit process is working under its optimal operating parameters such as temperature, mixing, ph, seeding etc
3 Elimination of physical and physiological bottlenecks in terms of pumping and even removing the effect of catabolite repressions or inhibitions
4 That the impact of any changes in upstream activities will affect the efficiency of the downstream activities. Any improvement or changes in upstream activities will affect downstream and not vice versa
5 That there must be allocation or allowances for changes even within this narrow range of optimized parameters



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PROBLEMS OF HIGH CELL DENSITY FERMENTATION

The microorganisms are important components in any microbial fermentation. The microorganisms in the fermentation process are either the agents of change which convert the substrate to valuable fermentation products or they themselves are the products sought in the fermentation process.
In either situation the objective of the fermentation process it selves is to obtain the highest number of microbes per unit volume. This trend is termed as achieving high cell density fermentation.
If we can achieve high cell density it is akin to saying that we have higher number of cells to carry out the transformation of substrate to products. More cells mean more “factory workers” to carry out the production. Thus more products will hopefully be produced over the period of fermentation process.
It should be noted although increasing the number of cells can increase the amount of fermentation products formed, that does not mean that the real efficiency of the fermentation process has increased in terms of fermentation yield by the unit cell. It is only the numbers of cells has increased leading to more conversions.
Only new strains or improved strains of producing microorganisms will increase the fermentation yield.
Logically, it is a good option to increase the fermentation product formation by increasing the number of microorganisms that will transform the fermentation substrates to fermentation products. However, this is not easily achieved in reality as there are many physical and physiological constraints that will try to prevent this objective.
The microbial cell is unique in its own right. During the process of fermentation not only it transforms the fermentation substrates to fermentation products, it will in the process of metabolism extract energy and carbon from the substrate to produce new biomass and undergo microbial growth.
In a way this would be ideal in achieving the high cell density fermentation, but such growth is not infinite and will ultimately slow down as in the typical sigmoid growth curve of the batch culture fermentation
Even before achieving this stationary phase the cells will it selves undergoes major changes in its metabolism or physiology will be of negative impact to the fermentation process and product formation
So how can we achieve the optimal high cell density fermentation? How shall we operate so that the fermentation of high cell density will be at optimum fermentation product formation in terms of economic and efficient control?
The whole idea of obtaining high cell density fermentation is ideal when:
1 You can have high density or number of microbial cells in the bioreactor
2 The high density protocol will not negatively affect the fermentation process or the products in terms of the quantity, quality and stability of products
3 That the fermentation process will be able to be carried out efficiently and economically in terms of substrate usage and efficient mass transfers
There are various strategies which could be applied in trying to obtain high density cell fermentation not only through feeding mode but also bioreactor configurations and physiological manipulations of the microbial populations





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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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