Fermentation kinetics

Microorganisms grow in a wide spectrum of physical and chemical environments, their growth and
other physiological activities are in fact a response to their physiochemical environment.
Fermentation kinetics describes growth and product formation by microorganisms- not only active
cell growth but also the activities of resting and dying cells, since many fermentation product of
cemmercial interest are produced after growth has stopped. This chapter is confined to kinetics in
batch culture. Kinetics of growth and product formation in continuous culture is considered in
chapter 7.

6.1 microbial

Microbial growth is usually characterized by the time required to double cell mass or cell number.
Mass doubling time may differ from cell doubling time because cell mass can increase without an
increase in cell number. However, if in a given environment the intervsl between cell mass or
number doublings is constant with me, the orgsanisme is growing at an exponential rate. Under such
conditions, growth is described by:

Specific growth rate

Thus equation 1 describes the increase in cell mass with time and equation 2 describes the increase
in cell number with time. Under most circumstances growth is measured by an increase in mass, so
µ will be used . the value µx is the volumetric growth rate (volumetric productivity ) in g/l-hr

Integration of equation 1 gives

In the specific growth rate is constant, equation 3 yields

Equation 4 may be solved for the case in which ∆t = td , that is , the time required for X2 = 2X1 , Then

From equation 4, it is seen that the specific growth rate is obtained from the slope of a plot of ln X
versus time

6.1.1 Bacterial Growth

There are four general patterns of microbial growth , exemplified by bacteria, yeast, molds, and
viruses.

Bacteria divide by fission as illustrated in figure 6.1 . during the growth cycle the cell doubles its mass
and the amount of all cell constituents. Prior to fission, cell wall and membrane are synthesized and
the parent cell divides into two identical daughter cells, each of which grows exactly as the original
cell. As a consequence, it is generally not possible to assign an age to a bacterium except within the
context of a single doubling. Thus reference to an old or young culture refers to the time spent in a
flask or on a shaker and not to the individual cells’ age.
One important exception to the concept of age occurs when an environmental change precludes the
need for a specific enzyme in the cell and the enzyme is no longer synthesized along with growth. As
the cell continues to divide , the existing enzyme may become exponentially diluted. As a
consequence , it is possible to specify culture age with reference to the enzyme level in the cell.

Doubling time for some bacteria is reported to be as fast as 15-20 minutes under optimal growth
conditions . however, 45-60 minutes is more typical

Identical daughter cells

Bacterial growth

Daughter cell

Bud scar

Mother cell

Yeast growth

Branching

Apical growth

Mycelial growth

6.1.2 yeast growth

Yeast belong to the fungi and commonly divide by budding. Exceptions to this include yeast that
grow by fission or by forming hyphae. Budding, as depicted in figure 6.1, involves the formation of a
bud on the mother cell. This bud grows until it approximates the size of the mother, at which point
the daughter cell separates. Unlike the division of bacteria, it is possible to physically distinguish
between the mother and the daughter cell because the mother cell retains a bud scar for each
daughter cell produced. Thus a growing population of yeast cells has a continually changing cell age
distribution. Under some growth conditions the buds do not separate from mother cell and long,
branched chains of cells (pseudomycelia) will form. Under optimal conditions yeast may divide in as
little as 45 minutes, however , 90-120 minutes is more typical.

6.1.3 mold growth

Another type of fungi are the molds, which characteristically grow by chain elongation and
branching as illustrated in figure 6.1. growth proceeds from the tip of the mycelium (apical growth)
by forming septa between the cells. Once a cell is formed it retains its integrity and has an age with
respect to the rest of its neighbors. Depending on the physicochemical environment, the mycelium
may be long and diffuse, short and highly branched, or a mixture of the two. When grown on a
surface, the mycelia may become intertwined and form thick mats. While in submerged culture the
mycelia may exist as diffuse mycelia or may form pellets with diameters ranging from 0,1-10 mm.
The mats and pellets are extremely important to the growth of molds since they will in turn affect
the local physiochemical environment of the individual mat or pellet cells. This is illustrated in figure
6.2 by the nutrient concentration profile in a pellet. Since metabolic reaction are dependent on
nutrient concentration, some pellet cells may often have reduced or otherwise altered rates of
metabolism.

It is very difficult to follow growth with respect to cell number in molds because the cells are not
easily separated. For this reason increases in mass are used to monitor growth. The age distribution
and the morphology of molds are both extremely important to the kinetics of fermentation
processes. In some processes only old cells produce the product , while in other processes young
cells are required. Typically , molds have optimal mass doubling times of 4-8 hours although some
are reported to double in as little as 60-90 minutes.

Actinomyces and streptomyces are classes of organisms closely related to bacteria that are
extremely important industrially . both classes also grow as mycelial organisms.

Nutrient and product concentration profiles in a mycelial pellet, if product is synthesized in the
center of the pellet the profile will look like (a) if the cells in the center are dead or nutrient starved,
the product profile may appear as (b)

6.1.4 virus growth

Microbial viruses or phage, do not follow the normal growth patterns. They require a host organism,
which may be a bacterium, yeast, mold, or higher from of cell. Viruses are structurally simple
consisting of genetic material (either DNA or RNA) usually incorporated into a protein sheath. They
may exist as single virus particles or become incorporated into the genome of the host cell. To
multiply, however , they must infect a cell and utilize the cell’s protein and nucleic acid synthesizing
machinery to produce new viral material . as a result, a single virus inside a cell may be replicated
50-300 times before the cell bursts and releases the newly formed viruses. Thus growth is
exponential but the exponent is much higher than two.

6.2 microbial growth: a chemical description

Growth may be looked at as a series of chemical reactions leading to the synthesis of cell mass. The
generalized stoichiometric formula for growth may be written as:

Where A, B, D and Q are the moles of the respective compounds, CaHbOc is a generalized carbon-
energy source, and M is the moles of a cell unit (CαHβOγNδ) . for instance a typical yeast may be
represented by C6H11NO3, thus the organic fraction of the cell has a unit molecular weight of 145. If
the cell is 90% organic and 10% ash, the overall formula weight of a unit cell is 145/0,9 = 161

From this stoichiometric relationship, one can see that the ratio of cell mass formed per mass of
substrate consumed (M/A) , referred to as the cell yield, is dependent on the efficiency of carbon
source utilization for incorporation into cell mass and for combustion to carbon dioxide and water to
obtain energy for growth. Furthermore, from this balance it is possible to devise a relationship
between the yield of cells on the carbon substrate and the requirement for oxygen. Mateles (1971)
derived the following useful expression for this purpose.

Figure 6.3 Oxygen requirement versus cell yield for microorganism grown on various substrates . cell
composition data are: O , 0,19; C, 0,53 ; N, 0,12; H, 0,07 for bacteria and C, 0,47; N, 0,075; O, 0,31 ;
H, 0,065 for yeast

Where C , H, and O are the number of atoms per substrate molecule and C’, O’ , N’ and H’ are the
fraction of C, O, N and H in the cell, Y is the cell yield coefficient (g cell/ g substrate) for the carbon
source , and Mw is the molecular weight of the carbon source .

From this expression, it is clear that the oxygen demand is inversely proportional to the cell yield,
which is a measure of the efficiency of conversion of the carbon source to cell mass. This relationship
is shown graphically in figure 6.3 for growth on several substrates.

6.3 methods for measuring microbial growth

A wide variety of methods are available for measuring microbial growth, yet no single procedure is
applicable in all situations. The method of choice depends on the measurement’s objective and on the
available techniques usefulness. In many casses , especially involving commercial fermentation , the
media for growth and product formation are so complex (solomons,1969) that direct methods for
estimating cell mass or number are not usable and a means for indirect assessment of cell mass is
necessary (calam , 1972). However , no matter what method is used, considerable care is required in
interpreting the results.

6.3.1 Measurement of cell number

Microscopic count. The most direct way to determine total cell number is by direct microscopic count.
This is done with the aid of a petroff-housser slide or a hemocytometer. Both of these devices are
microscope slides having a depression of known depth into which a cell sample is placed. A calibrated
grid is placed over the chamber and the number of cells per grid square are counted by microscope.
Usually 20 or more grid squares are counted and averaged. In order to distinguish between dead and
live cells, it is necessary to stain with viable stains sush as methylene blue or trypan blue which stain
only dead cells. In tjis manner, both total and viable counts can be made. A drawback of this
technique for bacteria is their small size and the shallow depth of field in the microscope when used at
high magnification. Examination of yeasts and molds is complicated by their not forming discrete
cells.

Viable plate count. Cell number is frequently mesured by the viable plate count. This method employs
petri dishes with agar containing an appropriate growth medium. The agar is spread with diluted
samples of microorganisms keeping each sample separated from its neighbor. These surface cultures
are incubated for 24 or more hours until discrete colonies are formed, each derived from a colony
forming unit (CFU) . it is important to distinguish a CFU from a single cell. If cells tend to aggregate,
form chains, pseudomycelia, or true mycelia, it is difficult , or impossible, to separate the units, and
one CFU may actually be more than one cell. This technique is most useful for bacteria and yeast and
less useful for molds. Care is needed also with many yeasts because they may form pseudomycelia or
true mycelia when grown under certain conditions.
When performing viable counts it is necessary to remember that viability is defined operationally as
the ability to grow in a specific environment. Therefor, care is required in the selection of an
appropriate medium for viable counts since some media support growth better than others. This is
especially true when a population contains dying or mutating cells.

Slide culture. A modification of the viable plate count is the slide-culture technique described in detail
by postgate, et al.(1961). This procedure employs an agar –gel medium placed in a small ring
mounted on a microscope slide. After plating cells on this miniature culture dish, the wholw slide is
placed in an incubator. After three to four doubling times, the slide is examined with a microscope
and the total number of microcolonies is divided by the total number of colonies plus nondividing
cells to yield the fraction of viable cells. This procedure has the same constraints as the total plate
count but is faster in that only a few cell doubling times are required before counting (3-4 hours rather
than 24)

Coulter counter . A sophisticated method for determining total cell count utilizes a coulter counter.
(gregg and steidler, 1965). A schematic of this device is shown in figure 6.4. a diluted culture sample
is placed in the liquid reservoir containing an electrolyte, and a vacuum is applied to the inner tube to
pull a predetermined volume of liquid through the small orifice. An electrical potential is applied
across the electrodes, and as cells pass through the orifice, the electrical resistance increases and
causes pulses in current. These pulses are than counted. In addition, the magnitude, of the resistance
change is proportional to the cell’s size, so by using a pulse height analyzer , the cell size distribution
may also be measured. A variery of orifice sizes are available for the coulter counter , and the smallest
size (30µ) must be used for bacteria which are typically 1-3 µ in diameter. This orifice is easily
plugged by dust particles or clumps of cells. Larger cells such as yeast, protozoa, or tissue cells can be
counted more successfully with larger orifices. Not surprisingly , the technique is difficult to use with
organisms in chains and is useless with mycelial organisms.

Nepholometry . total cell or particle number may be determined by nepholometry. This technique
employs a light source at a right angel to a phototube. As the light passes through the liquid sample ,
the phototube measures the scattered light. This procedur is useful for dilute cell and particle
suspensions.

6.3.2 Measurement of cell mass : direct methods

Cell dry weight. The most common method for measuring total cell mass is to obtain the dry cell
weight. Samples of whole culture broth are collected , centrifuged, washed with buffer or water, and
then dried at 80o C for 24 hours or at 110o C for 8 hours. For cells grown in a solids-free medium, this
technique is generally applicable. However, if noncellular solids such as calcium carbonate, molasses
solids, corn steep liquor, cellulose, or soy bean meal are present , they are not remove in the washing
and as a consequence contribute to the final dry weight. When calcium carbonate is the only insoluble
material, it is posible to use an acid wash to dissolve the salt and remove it from the cells prior to
drying. Even when noncellular solids are present, if the cells’s weight is much greater than the
impurities’ weight, dry cell weight is still useful although not precise.
Turbidity. The optical density or turbidity of a culture broth is proportional to the cell mass; this
method provides a simple , fast and inexpensive way to estimate cell mass on a routine basis. The
change in light intensity It through a distance L is given by:

Where ε is the molar absorption coefficient and C is the concentration, integrated of equation 8 yields

Where ε’ is the molar extinction coefficient. Therefore, a plot of ln (It/I0) versus the concentration c
will yield a straight line with a slope –εL. L is usually chosen as 1 cm (It/I0) x 100 is defined as
percent transmittance T and absorbance is equal to log10(1/T). Rearranging equation 8 to give the
absorbance A

Therefor, a plot of absorbance on linear coordinates versus concentration yields a straight line.

The turbidity of a cell suspension is usually with light at a wavelength between 600-700 nm when
using a spectrophotometer or with light through a red filter when using a colorimeter, it is important
not to go outside the linear range of the assay. Most spectrophotometers are linear up to two or more
absorbance units; one absorbance unit is approximately one-half to one gram of dry cell weight. Klett-
summerson colorimeters are linear up to about 125 klett units and typically 250-300 klett units equals
one gram of dry cell weight. It is important when using turbidity as a measure of cell mass to be
certain that fermentation product or medium components do not absorb light at the wavelenght used.
This technique is not useful if substansial amounts of noncellular solids are in the medium . when
turbidity is used for estimates of mycelial organisms, it is sometimes necessary to homogenize the
sample in a waring blender before analysis.

6.3.3 estimation of cell mass : indirect methods

It is apparent that the nature of the organism and the medium components greatly affect the ease with
which cell mass may be measured. In many cases-such as mold or other mycelial fermentations to
resort to indirect methods to assess growth. The interpretation of indirect methods for measuring cell
mass is facilitated by keeping in mind the overall stoichiometry for growth and product formation ,
which may be written in a general form.

C-source + N-source + phosphate

From this equation it is apparent that methods for measuring nutrient consumption or product
formation may be related to microbial growth.

Cell components. One useful method to assess cell concentration is to measure a macronolecular cell
component . however, because the proportion of these materials in a cell can change with time, care is
needed in interpreting the result. . during exponential growth with a constant growth rate , the cell
composition is constant , as shown in figure 6.5 . during the early and late part of the growth cell
composition changes. Proteint content is fairly constant , while RNA varies dramatically with growth
rate. DNA, probably the most constant , is not usually found in noncellular materials added as
nutrient, analysis for DNA is laborious (burton, 1957) but, in complex heterogeneous media, where
specificity is necessary for cell mass measurement , it is useful.

Analysis of cellular proteint content deserves some mention. The most common methods are biuret
(stickland, 1951), folin (Lowery et al., 1951), kjehdahl nitrogen, and total amino acid analysis. Each
method gives a different value for cellular protein since each reaction is different. However, The
biuret reaction is quite useful since it primarily measures peptide bonds and there is little or no
interference from other nitrogen-containing compounds. Furthermore, it is a simple and rapid
procedure.

Elemental analysis of the cells can also be employed. The most common technique is total nitrogen
analysis by the kjehdahl method. Carbon, hydrogen, oxygen, and nitrogen analysis is also possible
with an elemental analyzer. The latter, however,require expensive equipment and in all cases it is
necessary to separate the cells from other medium components. If the elemental composition is
determined it can be related to growth via the sto:chiometry presented in equation 6.

Nutrient Uptake. An alternative approach to cellular analysis is to measure the removal of an essetial
nutrient from the medium. It is desirable to choose a nutrient not likely to be used to synthesize a
metabolic product. Phosphate,suliate, or magnesium may be likely candidates. When cell mass is the
major product, the carbon energy source or oxygen are particularly useful for this purpose,

Product formation, frequently, products of cell growth are useful for the estimation of growth as well
as being the primary objective of the fermentation. A common product , carbon dioxide, resulting
from the oxidation of the carbon energy source, is readily measured with an infrared gas anlyzer, and
can be related stoichiometrically to growth. Many anaerobic fermentations are characterized by
incomplete oxidation of the carbon energy source and the accumulation of product s such as lactic
acid or ethanol. Care is needed, however, in relating these products to growth in that their formation
may be both growth and nongrowth associated .

One product that is quite general is the hydrogen ion. If ammonium ion is used for the hydrogen
source, then for each mole ammonium consumed, a hydrogen ion is produced. This must be
neutralized to maintain constant pH, and as a consequence the amount of alkali added is proportional
to growth. If nitrate is used for nitrogen, one must monitor addition of acid for neutralization.

Heat evolution . a universal product of microbial growth is heat . microorganisms do not violate the
laws of thermodynamics and as growth proceeds, heat is evolved. The heat of combustion of
microorganisms is fairly constant with a typical value of 5 kcal/g and the heat evolution will depend
upon the efficiency with which the carbon energy source is utilized. This point is discusses in detail
later in this chapter. Suffice it to say here heat is stoichiometrically related to growth and product
formation.

Several methods of heat mesurement exist, but two most suitable for use with fermentors are an
energy balance on the cooling water and the technique of dynamic calorimetry (cooney et al., 1968)

When an energy balance is written for a fermentation , the following equation is obtained

Where Qacc is the net heat accumulation

QF is the heat of fermentation

Qag is the heat from agitation

Qevap is heat from evaporation

Qsen is the sensible heat in the air stream

Qsurr is the heat dissipated to the surroundings through the fermentor walls
Qsen is usually much less than QF and may be neglected. Qevap can be eliminated by using air saturated
with water at the temperature of the fermentation, or it can be calculated from the wet and dry bulb
temperatures of the entering and exiting gas. Qag + Qsurr can be measured prior to inoculation of the
culture at several values of agitation speed and airflow. With the information given here, the hear of
fermentation may be continuously measured.

Dynamic calorimtery employs the whole fermentor as an adiabatic calorimeter. To measure the heat
of fermentation , temperature control is shut off and the temperature is allowed to rise by 0,5-1,0oC.
The heat accumulation is then estimated from the slope the temperature time curve (∆T/∆t) and the
total heat capacity of the system C’p in kcal/oC as shown in equation

An alternative approach is to measure the flow rate (w) , inlet(Tin) and exit (Tout) temperatures of the
cooling water . this is difficult to do in small fermentors and is more useful when the surface to
volume ratio of the vessel is small. The energy balance on the cooling water may be written

Where Cp is the specific heat of water.

Typical values for each of the heat terms in fermentation are presented in table 6.1 . thus early in the
fermentation substansial error may occur, but as the rate of heat evolution increase the method
becomes increasingly useful