Thursday, 7 December 2017

FED BATCH KINETICS

FED BATCH KINETICS


Fed Batch as the name indicates means batch cultures which need to be fed continuously, with medium without removing culture fluid. The culture broth is harvested usually only at the end of the operational period, either fully or partially (the remainder serving as the inoculum for the next repeated run). This process may be repeated (repeated fed-batch) a number of times if the cells are fully viable and productive. Thus, there are one or more feed streams but no effluent during the course of operation. Sources of carbon, nitrogen, phosphates, nutrients, precursors, or inducers are fed either intermittently or continuously into the culture by manipulating the feed rates during the run. The products are harvested only at the end of the run. Therefore, the culture volume increases during the course of operation until the volume is full. Thereafter, a batch mode of operation is used to attain the final results. Thus, the fed-batch culture is a dynamic operation. By manipulating the feed rates, the concentrations of limiting nutrients in the culture can be manipulated either to remain at a constant level or to follow a predetermined optimal profile until the culture volume reaches the maximum, and then a batch mode is used to provide a final touch. In so doing, the concentration of the desired product or the yield of product at the end of the run is maximized. This type of operation was first called a fed-batch culture or fed-batch fermentation. 
The fed-batch culture has been practiced since the early 1900s, when it was recognized in yeast production from malt wort that the malt concentration in the medium had to be kept low enough to suppress alcohol formation and maximize the yield of yeast cells. High malt concentration would accelerate the cell growth, which in turn would cause anaerobic conditions that favored ethanol formation and lowered the yield of yeast cells. Additional wort was added at a rate that was always less than the rate at which the yeast cells could use it. Intermittent or incremental feeding of nutrients to an initially dilute medium was introduced thereafter in large-scale yeast production to improve the yeast yields while obviating the production of ethanol.4 However, there is some speculation that a small amount of ethanol may be necessary to ensure the quality of the baker’s yeasts.
The oldest and first well-known industrial application of a fed-batch operation was introduced after the end of World War I. It was the yeast cell production in which sugar (glucose) was added incrementally during the course of fermentation to maintain a low sugar concentration to suppress alcohol formation. The manufacture of yeast by fed-batch culture has gone through a series of improvements and is an industrially important fed-batch process. This process was historically followed by penicillin fermentation, in which the energy source (e.g., glucose) and precursors (e.g., phenyl acetic acid) were added incrementally during the course of fermentation8 to improve penicillin production. Prior to this practice, a slowly metabolized but more expensive substrate, lactose, was used in place of glucose in a batch culture. Oversupply of a carbon source resulted in more mycelial growth and low penicillin formation, while undersupply resulted in slower mycelial growth and, eventually, slower penicillin formation.
The primary candidates in the list of compounds that may be fed during the course of the operation include the limiting substrate, inducers, precursors, a carbon source, a nitrogen source, a phosphate source, inducers, and other nutrient sources. The feeding patterns are open loop or feedback controlled to maintain some key variables at constant optimum values such as the specific growth rate, respiratory quotient, pH, partial pressure of carbon dioxide, dissolved oxygen, substrate concentration, and some metabolite concentrations. The optimum feed rates sometimes require keeping these parameters to follow certain optimum profiles rather than keeping them at constant values. To maximize the cell formation rate for the case of constant cell mass yield, it is obvious that the substrate concentration should be maintained at the value that maximizes the specific growth rate, Sm, until the reactor is full. Therefore, it is also obvious that the initial substrate concentration should be Sm, that is, S(0) = Sm, and that the substrate concentration should be maintained at Sm throughout the course of fermentation. This will lead to the maximum cell concentration at the end of the run. To achieve this, the feed rate must be regulated properly to hold the substrate concentration constant at Sm. If it is not possible to set the initial substrate concentration to Sm for one reason or another, the substrate concentration should be brought to this value as soon as possible by applying at the beginning the maximum substrate feed rate (S(0) < Sm) or a batch period (S(0) > Sm) and then regulating thereafter to maintain the substrate concentration to remain at Sm until the fermentor is full. Once the fermentor is full, it is run in a batch mode to reduce the substrate concentration to a desired level, and the cells containing the product are harvested. This is the basis for the simplest case of a fed-batch culture.


 Initial establishment of fed batch is in BATCH mode and then the feeding is done by applying one of several strategies. 

lets see the strategies.... Quite interesting..uhmm

1.Use the same medium which you have used to establish the batch culture and add it. what will happen? increase in volume.. right???

2.Take the solution of limiting substrate; keeping concentration same as it was in initial medium & add it... again . tell me the result.... increase in volume... yes ! exactly..

Fed batch using above two strategies will be described as Variable Volume Fed Batch Culture

3.In this strategy concentrated solution of limiting substrate is to be added but at a rate less than no.1 & 2 strategy with the result is increase in volume.

Fed Batch employing above strategy is called as Intermediate Fed Batch Culture

4. This time a very concentrated solution of limiting substrate is added but at a rate less than strategy 1,2&3, resulting in overall increase in specific volume.

Fed Batch utilising above strategy is called as Fixed Volume Fed Batch Culture


So now we have an idea that what the heck is this fed batch strategy ..lets move on to the devilish part that is kinetics!!!! Start with Variable Volume Fed Batch culture

Variable Volume Fed Batch Culture

Pirt the king of kinetics ( That's what i call him......) , Dunn & Mor has developed this culture. I'm discussing Pirt's kinetics only. He said consider a batch culture in which growth is limited by concentration of one substrate; so whats the equation if we want to describe the biomass at any point of time::::

Equation for fed batch kinetics (variable volume)






Thus from above equation it may be concluded that input of substrate is compensated by substrate consumption. Thus ds/dt =0
Total biomass (X) concentration increases with time but cell concentration (x) remains constant, i.e. dx/dt = 0 and we can now write 
μ= D
This situation is quasi steady state
(P.S. ::: please go back to basics of kinetics if you have trouble following above equation.. )

The difference between steady state in chemostat and quasi steady statein fed batch is that μ is constant in chemostat and it decreases in fed batch.


Fixed volume in next blog ....till then understand it and let me know if youy face any issues


CREDITS::: Whitaker, Pirt, Cambridge.













Thursday, 14 August 2014

Phagocytosis


Phagocyosis is the process of engulfment and digestion of microorganisms performed by macrophages & microphages.
The macrophages:
These cells originate from bone marrow promonocytes; after circulating in blood for few hours they migrate to tissues and constitute the mononuclear phagocyte system. They have been given specific names in accordance with their residing tissues; in lungs ; alveolar macrophages, in  liver ; kupffer cells, microglial cells in brain, mesangial cells in kidney and in bone as osteoclasts.
Macrophages are capable of ingesting and digesting exogeneous antigens such a s whole microorganisms and insoluble particles and endogeneous matter such as injured or dead cells. Macrophages act by process of chemotaxis in which they get attract towards variety of substances. In the next step antigen adheres to the cell membrane of macrophage. Adherence induces the protrusions called Pseudopodia to extend around the attached antigen. Fusion of pseudopodia encloses the antigen with in membrane bound structure called Phagosome which then enters the phagocytic processing pathway. Here Phagosome fuses with the lysosome and makes phagolyosome. Lysome has hydrolytic enzymes which digest the ingested material. The digested contents are then released through the process of exocytosis.

It has been found that phagocytosis of antigen can be enhances many times in the presence of specific antibody; so antibody functions as opsonin; a molecule that binds to antigen and macrophage and enhance the process of engulfment. This whole process whereby antigens are rendered more susceptible to phagocytosis is called as opsonisation.

for further reads:::

kuby's immunology