Etiket Arşivleri: stationary phase

Microbial Kinetics and Substrate Utilization in Fermentation

Microbial Kinetics and Substrate utilization in Fermentation

Batch culture and Kinetics of Microbial growth in batch culture

After inoculation the growth rate of the cells gradually increases.

The cells grow at a constant, maximum, rate and this period is known as the log or exponential, phase.

Growth of a typical microbial culture in batch conditions

The rate of growth is directly proportional to cell concentration or biomass-

i.e.           dx/dt     α      X                        

               dx/dt   =  μX                        ———-1

 Where,

     X is the concentration of microbial biomass,

   t  is time, in hours

   μ  is the specific growth rate, in hours -1

On integration of equation (1) from t=0 to t=t ,we have:

                    xt = xo e μt                         ——— 2

Where,

Xo   is the original biomass concentration,

Xt  is the biomass concentration after the time     interval, t hours,

e is the base of the natural logarithm.

On taking natural logarithms of equation (2) we have :

         In Xt = In Xo + μt                 (3)

Therefore, a plot of the natural logarithm of biomass concentration against time should yield a straight line, the slope of which would equal to μ.

During the exponential phase nutrients are in  excess and the organism is growing at its maximum specific growth rate, ‘μmax  ‘ for the prevailing conditions.

Typical values of μmax for a range of microorganisms are given below in the Table.

From figure 2 it may be seen that over the zone A to B due to an increase in initial substrate concentration  gives a proportional increase in the biomass occur at stationary phase. This relation between increase in initial substrate concentration and proportional increase in the biomass may be described by equation:

                X = Y(SR – s)    ———(3)

Where,

  X –is the concentration of biomass produced,

  Y –is the yield factor (g biomass produced g-1 substrate consumed),

  SRis the initial substrate concentration, and

   s  -is the residual substrate concentration.

Thus, equation (3) may be used to predict the production of biomass from a certain amount of substrate

In Fig. 2:-

Over the zone A to B: s = 0; at the point of cessation of growth.

Over the zone C to D an increase in the initial substrate concentration does give a proportional increase in biomass due to the exhaustion of another substrate or the accumulation of toxic products

The decrease in growth rate and the cessation of growth due to the depletion of substrate, may be described by the relationship between μ and the residual growth limiting substrate.

This relationship is represented by a equation  given by Monod in1942 is know as Monod equation.

Based upon Michaelish-Menten kinetics.

According to Monad equation-

   μ  =   μmax . S /Ks + S               (4)

Where,

S  is residual substrate concentration,

Ks is substrate utilization constant, numerically equal

to substrate concentration when μ is half of μmax.

Ks  s a measure of the affinity of  the organism with substrate

It tell about the relationship between specific growth rate ‘μ’ and growth limiting substrate concentration ‘S’.

If the organism has a very high affinity for the limiting substrate (a low Ks value) the growth rate will not be affected until the substrate concentration has declined to a very low level. Thus, the deceleration phase for such a culture would be short.

However, if the organism has a low affinity for the substrate (a high Ks value) the growth rate will be deleteriously affected at a relatively high substrate concentration. Thus, the deceleration phase for such a culture would be relatively long.

The biomass concentration at the end of the exponential phase is at its highest  level. Therefore the decline in substrate concentration will be very rapid so that the time period during which the substrate concentration is close to Ks is very short.

The stationary phase in batch culture is that point where the growth rate has declined to zero. This phase is also known as the maximum population phase.

Kromatografik Yöntemler ve HPLC Yöntemi

 

Kromatografi Nedir?

Kromatografi, bir karışımda bulunan bileşenlerin birbirinden ayrılmasını gerçekleştiren ve bu sayede kalitatif ve kantitatif analizlerinin yapıldığı yöntemlerin genel adıdır. Bu yöntemlerde çalışma düzeneği temel olarak iki bileşenden oluşur. Bu bileşenlere sabit faz (stationary phase) ve hareketli faz ya da mobil faz (mobile phase) adı verilir. Mobil fazın içerisinde yer alan bileşenler, sabit faza ait dolgu maddesiyle etkileşmeleri sebebiyle, bir miktar tutulurlar. Bu tutulma, örnekteki farklı bileşenler için farklı miktarlarda olur. Böylece bileşenler sabit fazın sonlarına doğru, farklı hızlarda ilerledikleri için, birbirinden ayrılmış vaziyette sabit fazı farklı zamanlarda terkederler. Bu şekilde sabit fazdan çıkan bileşenlerin derişimleri uygun bir biçimde ölçülür ve zamana veya mobil fazın kullanılan hacmine karşı y-ekseninde işaretlenerek “kromatogram” denilen grafikler elde edilir.

Kromatografi’nin Temel Prensibi ve Tanımları

Mobil faz (mobile phase): Örnek Bileşenlerini, sabit faz (kolon) boyunca taşıyan, çeşitli fiziksel ve kimyasal özelliklere sahip çözelti veya çözücü karışımları. Kullanılacak mobil fazın seçiminde, analizi yapılacak örnek madde bileşenlerinin özellikleri, kullanılacak sabit faz ve dedektörün özellikleri vb. birçok parametreye dikkat edilmelidir.

Sabit faz (stationary phase): Mobil faz içerisinde gelen örneğe ait bileşenlerin etkileşime girdikleri ve belirli ölçüde alıkonuldukları fazdır. Kromatografi tekniğinin çeşidine göre tasarlanmış ve çok değişik materyallerden çok farklı ölçülerde imal edilmiş ve “kolon” olarak adlandırılmış sabit fazlar mevcuttur. Özellikle gaz ve sıvı kromatografileri için ticari boyutta oldukça fazla marka ve boyutta kolon üretimi yapılmaktadır. Sıvı kromatografisinin bir çeşidi olan yüksek performas sıvı kromatografisi (HPLC) uygulamalarında kullanılan kolonlar daha çok 30-300 mm uzunluğunda yaklaşık 5 mm iç çapında metalik boru şeklinde olup iç yüzeyleri çok değişik özelliklerde kaplama materyalleri ile kaplanarak, analizi yapılacak madde grupları için modifiye edilebilmektedir.

Alıkonma (retention): Mobil faz içerisinde gelen, analizi yapılacak maddeye ait bileşenlerin sabit faz ile etkileşime girerek belirli oranda tutulması daha doğrusu yavaşlatılması ve böylece daha geç olarak sabit fazı terk etmesi olayıdır. Bu özellikten yola çıkılarak, belirli sabit analitik koşullar altında, her kimyasal madde için parmak izi niteliği taşıyan alıkonma zamanı (retention time-tR) tanımı türetilmiştir. Bu kavram belirli sabit deneysel koşullarda analizi yapılan maddenin sabit fazı terketmesi için geçen süreyi göstermektedir.

Fundamentals of Liquid Chromatography (HPLC)

First, What is Liquid Chromatography?

•  Liquid chromatography is a separation technique that involves:

•  the placement (injection) of a small volume of liquid sample

•  into a tube packed with porous particles (stationary phase)

•  where individual components of the sample are transported along  the packed tube (column) by a liquid moved by gravity.

• The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. • The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount.  F Note: The modern form of liquid chromatography is now referred to   as “flash chromatography”


Chapter 5 ( Dr. Ali Coşkun DALGIÇ )

FE 462 BIOCHEMICAL ENGINEERING
Cell Kinetics and Fermenter Design
INTRODUCTION
§Understanding the growth kinetics of microbial, animal, or plant cells is important for the design and operation of fermentation systems employing them. Cell kinetics deals with the rate of cell growth and how it is affected by various chemical and physical conditions.
Unlike enzyme kinetics, cell kinetics is the result of numerous complicated networks of biochemical and chemical reactions and transport phenomena, which involves multiple phases and multicomponent systems.
The heterogeneous mixture of young and old cells is continuously changing and adapting itself in the media environment which is also continuously changing in physical and chemical conditions. As a result, accurate mathematical modeling of growth kinetics is impossible to achieve. Even with such a realistic model, this approach is usually useless because the model may contain many parameters which are impossible to determine
GROWTH CYCLE FOR BATCH CULTIVATION

1. Lag phase: The lag phase (or initial stationary, or latent) is an initial period of cultivation during which the change of cell number is zero or negligible. Even though the cell number does not increase, the cells may grow in size during this period.

The length of this lag period depends on many factors such as the type and age of the microorganisms, the size of the inoculum, and culture conditions.

2. Exponential phase: In unicellular organisms, the progressive doubling of cell number results in a continually increasing rate of growth in the population. A bacterial culture undergoing balanced growth mimics a first-order autocatalytic chemical reaction.

The rate of the cell population increase at any particular time is proportional to the number density of bacteria present at that time:

Factors affecting the specific growth rate

Substrate Concentration: One of the most widely employed expressions for the effect of substrate concentration on μ is the Monod equation, which is an empirical expression based on the form of equation normally associated with enzyme kinetics :

The value of Ks is equal to the concentration of nutrient when the specific growth rate is half of its maximum value μmax. According to the Monod equation, further increase in the nutrient concentration after μ reaches μmax does not affect the specific growth rate.

Factors affecting the specific growth rate

Product Concentration: As cells grow they produce metabolic byproducts which can accumulate in the medium. The growth of microorganisms is usually inhibited by these products, whose effect can be added to the Monod equation as follows:

3. Stationary phase: The growth of microbial populations is normally limited either by the exhaustion of available nutrients or by the accumulation of toxic products of metabolism. As a consequence, the rate of growth declines and growth eventually stops. At this point a culture is said to be in the stationary phase.

4. Death phase: The stationary phase is usually followed by a death phase in which the organisms in the population die. Death occurs either because of the depletion of the cellular reserves of energy, or the accumulation of toxic products. Like growth, death is an exponential function. In some cases, the organisms not only die but also disintegrate, a process called lysis.

Modeling of the Bacterial Growth Curve

M. H. ZWIETERING,* I. JONGENBURGER, F. M. ROMBOUTS, AND K. VAN ‘T RIET

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1990, p. 1875-1881

Bioreactors

Stirred Tank Fermenters
A bioreactor is a device within which biochemical transformations are caused by the action of enzymes or living cells. The bioreactor is frequently called a fermenter whether the transformation is carried out by living cells or in vivo cellular components (enzymes).

For a large-scale operation the stirred-tank fermenter (STF) is the most widely used design in industrial fermentation. It can be employed for both aerobic or anaerobic fermentation of a wide range of cells including microbial, animal, and plant cells.

Stirred Tank
Fermenters
Kinetics of Substrate Utilization, Product Formation, and Biomass Production in Cell Cultures
It is difficult to obtain useful kinetic information on microbial populations from reactors that have spatially no uniform conditions. Hence it is desirable to study kinetics in reactors that are well mixed.

Ideal Batch Reactor

Many biochemical processes involve batch growth of cell populations. After seeding a liquid medium with an inoculum of living cells, nothing (except possibly some gas) is added to the culture or removed from it as growth proceeds. Typically in such reactor, the concentrations of nutrients cells, and products vary with time as growth proceeds.
A material balance on moles of component i;

The Ideal Continuous Flow Stirred Tank Reactor (CSTR)
The diagram of this process is shown in fig.2, which is a schematic diagram of completely mixed stirred tank reactor. Such configurations for cultivation of cells are frequently called chemostats.

The Ideal Continuous Flow Stirred Tank Reactor (CSTR)

Productivity of CSTF
MULTIPLE FERMENTERS CONNECTED IN SERIES
A question arises frequently whether it may be more efficient to use multiple fermenters connected in series instead of one large fermenter. Choosing the optimum fermenter system for maximum productivity depends on the shape of the l/rx versus Cx curve and the process requirement, such as the final conversion.

Cell Recycling
For the continuous operation of a PFF or CSTF, cells are discharged with the outlet stream which limits the productivity of fermenters. The productivity can be improved by recycling the cells from the outlet stream to the fermenter.

Alternative Fermenters
Many alternative fermenters have been proposed and tested. These fermenters were designed to improve either the disadvantages of the stirred tank fermenter-high power consumption and shear damage, or to meet a specific requirement of a certain fermentation process, such as better aeration, effective heat removal, cell separation or retention, immobilization of cells, the reduction of equipment and operating costs for inexpensive bulk products, and unusually large designs.