GROWTH AND CULTIVATION OF BIOOBJECTS - V. M. Samygin - 2016

CHAPTER 5. GROWTH AND REPRODUCTION OF MICROORGANISMS

The continuous synthesis of cellular components carried out by microorganisms stimulates Cell growth and subsequent reproduction. Cell growth is The process of increasing the size, volume, and mass of each individual Organism during its ontogenetic development, driven by the synthesis of cellular material. Growth begins after Cell Division; The Cell quickly reaches maturity and then proceeds to reproduction or enters a resting stage. Growth is characteristic of all groups of microorganisms, except Viruses, and is studied using isolated Cells.

Microbial reproduction is a self-Replication process that ensures the preservation of a species. The modes and rate of reproduction are determined by the microbe's genome and the compatibility of environmental conditions with its genetic requirements. Modes of reproduction among microorganisms are diverse: binary fission in most Bacteria; budding, spore formation, and Sexual reproduction in Fungi and Yeasts; and specialized reproductive mechanisms in Protozoa (multiple fission, sexual processes, etc.).

During binary fission—initiated by the bacterial circular DNA attached to a specific site of the cytoplasmic membrane—two equivalent daughter cells are formed from a single parent cell without prior exchange of Genetic information. METABOLISM/36.html">DNA replication begins at a specific phase of growth under the control of regulator and replicator genes. The newly synthesized DNA attaches to an adjacent site of the cytoplasmic membrane. A constriction (in gram-negative bacteria) or a septum (in gram-positive bacteria) forms strictly along the equator between the attachment sites of the original and newly synthesized DNA. Following division, the daughter cells separate or form colonial arrangements such as chains, clusters, packets, etc.

Cell growth should be distinguished from population growth, which is the irreversible increase in living matter (biomass) driven by an increase in cell mass and cell number per unit volume of the nutrient medium. The quantitative study of growth can be presented more informatively and precisely by analyzing various growth parameters, such as specific growth rate, lag phase, economic coefficient, metabolic coefficient, biomass yield, etc.

5.1. Microbial growth Curve in Batch Culture

The following conditions are required for biomass growth in culture:

a) viability of the inoculum;

b) presence of an energy source;

c) addition of nutritional supplements containing all components necessary for biomass synthesis;

d) absence of growth-inhibiting substances in the medium;

e) maintenance of suitable physicochemical conditions in the medium.

When bacteria are introduced into a nutrient medium, they typically grow until

the concentration of one of the essential environmental components drops to a minimum, after which growth ceases. If no nutrients are added and no metabolic end products are removed during this period, a batch culture (a population of cells in a confined living space) is obtained. Growth in such a “closed system” obeys regularities that apply to both unicellular and Multicellular Organisms.

The curve describing the logarithm of the Number of viable cells as a function of time is called the growth curve. A typical growth curve is S-shaped (sigmoid) and allows the identification of several sequential growth phases: the initial (or lag) phase, the acceleration phase, the exponential (or logarithmic) phase, the stationary phase, the deceleration phase, and the death phase (Fig. 5).

Class="center">Fig. 5. Growth curve of microorganisms in batch culture: I - lag phase; II - acceleration phase; III - exponential growth phase; IV - deceleration phase; V - stationary phase; VI - culture death phase

Initial phase. This phase spans from the moment of inoculation until the culture reaches its maximum growth rate. The duration of this phase depends mainly on the volume and age of the inoculum, previous cultivation conditions, and the suitability of the selected medium for growth. Introducing a small volume of inoculum into a large volume of fresh medium can lead to the diffusion out of the cells of Vitamins, Cofactors, and ions necessary to maintain The activity of many intracellular Enzymes. As the age of the inoculum increases, or when cells are transferred from a poor medium to a rich one, the lag phase lengthens.

If an old culture (in the stationary growth phase) is used as the inoculum, the cells must first adapt to the new conditions by synthesizing RNA, forming Ribosomes, and producing new enzymes. If the energy and carbon sources in the new medium differ from those in the previous culture, adaptation to the new conditions may involve synthesizing new enzymes that were not previously needed and thus were not produced. The formation of these new, adaptive enzymes is induced by the new substrate. The time required to synthesize an optimal amount of enzymes can range from 10 minutes to several hours. In some species, enzyme induction for utilizing new carbon and Energy Sources does not occur as long as even trace amounts of the original carbon and energy sources remain in the medium.

A clear example of substrate influence on enzyme synthesis is diauxie. This phenomenon of biphasic growth, or a double growth cycle, is observed in media containing a mixture of nutrients. For instance, in a mixture of glucose and lactose, E. coli consumes glucose first. Glucose induces the synthesis of enzymes required for its utilization while simultaneously repressing (suppressing) the synthesis of enzymes needed for lactose utilization. These latter enzymes are produced only after all the glucose has been depleted. Such regulatory processes explain the presence of two initial phases (Fig. 6).

Fig. 6. Growth of E. coli in a medium with different sugars (diauxie)

When evaluating growth, certain deviations from the ideal growth curve may be observed. If bacterial mass is measured via turbidity, the onset of the stationary phase is often preceded by a slight decrease in optical density. This can be an artifact caused by a changing ratio between biomass and optical density. Another deviation is a more rapid increase in cell number During the first doubling, which may be due to synchronous division of the unicellular population. A third variation is diauxie.

The quantitative change in the composition of a bacterial cell during the initial growth phase most prominently affects RNA: its content increases 8- to 12-fold. This indicates the active involvement of RNA and ribosomes (composed of RNA and Protein) in the synthesis of enzyme Proteins.

The lag phase may be associated with the inactivation of certain inhibitors present in the medium. Trace Elements sometimes act as such inhibitors. Their toxic effect is linked to the potential formation of complexes between Amino Acids and Metal Ions present in the nutrient medium. Organic acids also exhibit toxic properties, though their toxicity decreases as the pH rises. The substrate itself can act as an inhibitor; in this case, the lag phase will depend on the time required to reduce the Substrate Concentration to levels that allow for the maximum growth rate. Metabolic products within the inoculum can likewise exert an inhibitory effect on growth. For example, certain representatives of Lactobacillaceae, when transferred from an anaerobic to an aerobic environment, may accumulate hydrogen peroxide, which is a potent growth inhibitor. Hydrogen peroxide induces the formation of peroxidase, an enzyme that decomposes hydrogen peroxide. The lag phase caused by hydrogen peroxide can increase with a larger inoculum size, as The production of hydrogen peroxide is proportional to cell concentration. If the inoculum consists of spores, the lag phase preceding vegetative growth corresponds to the spore germination time.

Thus, various factors can cause growth delays. To avoid or minimize the lag phase, the inoculum should be taken from a culture at the end of its logarithmic phase, using conditions as close as possible to the transfer medium. Generally, the inoculum size should be kept as large as feasible.

The exponential (logarithmic) phase of growth is characterized by a constant maximum rate of cell division, which depends on the bacterial species and the nutrient medium. Enterobacteria divide every 15–30 min, many soil species require 60–150 min, and for Nitrobacter, it can even take 5–10 h. Cell size and protein content in many bacteria also remain constant during the exponential phase. In other words, the bacterial culture consists of "standard cells." If the cell count, protein content, and dry biomass all increase at the same rate, culture growth can be monitored using any of these indicators.

Since the cell division rate remains relatively constant during the exponential phase, this period is most convenient for determining the cell division rate (growth rate) and studying the Influence of Environmental factors (such as pH, redox potential, Temperature, and aeration). Additionally, this phase is used to evaluate the suitability of various substrates, as well as to monitor increases in cell number or the turbidity (optical density) of the cell suspension.

The stationary phase begins when the cell count stops increasing. The growth rate depends on substrate concentration; as this concentration decreases—even before the substrate is fully depleted—the growth rate starts to drop. Consequently, the transition from the exponential phase to the stationary phase occurs gradually. Growth may slow down not only due to nutrient depletion but also because of high bacterial population density, low partial pressure of O₂, or the accumulation of toxic Metabolic waste products. All these factors trigger the shift to the stationary phase.

During the early stationary phase, bacterial cell size reaches its minimum. In the late stationary phase (as well as the death phase), distorted or swollen cells, known as "involution forms," are frequently observed. This is typically related either to cell Damage caused by lytic enzymes targeting cell walls or Plasma Membranes, or to poor Regulation of cellular component synthesis. In Gram-positive bacteria, cells in the stationary phase often lose their Gram-staining ability. Bacterial resistance to Physical and Chemical stresses (such as hypotonic environments or sudden temperature shifts) is higher in the stationary phase than in the logarithmic phase. For certain bacteria, spore formation is also characteristic of the stationary phase.

Toward the end of the logarithmic phase, the synthesis of primary metabolites takes place—these are products essential for growth and survival. In the stationary phase, secondary metabolites are synthesized; these are products not required for growth or vital for survival, yet they perform useful Functions and often protect the cell against competing microorganisms or inhibit their growth (e.g., Antibiotics). The amount of biomass achieved in the stationary phase is referred to as the yield. The yield depends on The Nature and quantity of the nutrients used, as well as the cultivation conditions.

Death phase. Cell death is largely a result of the exhaustion of intracellular energy reserves. Cells stop dividing, die, and undergo lysis. The number of viable cells drops sharply. The rate of bacterial death varies depending on environmental conditions and the organism's specific traits.

5.2. Parameters of the Growth Curve

The main growth parameters of a simple batch culture of microorganisms (specific growth rate, generation time, number of cell divisions) are determined during the exponential phase, whereas the maximum concentration of viable cells in the population is typical of the stationary phase.

5.2.1. Specific Growth Rate

If the necessary conditions for biomass growth in a culture are met, it is assumed that during the logarithmic growth phase, over an infinitesimally small time interval dt, the increase in biomass dx must be proportional to the biomass amount x and the time interval, that is,

dx = μx dt, (1)

whence

dx/dt = μx (2)

The differential ratio dx/dt expresses the population growth rate. The parameter μ, which denotes the growth rate per unit of biomass (1/x)(dx/dt), is called the specific growth rate and is measured in reciprocal time units (1/t). This parameter is analogous to compound interest. For instance, a specific growth rate of 0.1 h^-1 is equivalent to a rate of 10% per hour.

If μ is constant, integrating equation (2) yields

ln x = ln x₀ + μt, (3)

where x₀ is the biomass at the initial time point t=0. A graph of ln x versus time will appear as a straight line with a slope of μ.

It follows from equation (3) that

ln (x/ x₀) = μt (4)

then

μ = ln (x/x₀): t, (5)

and

x = x₀ e μt (6)

Growth that follows this law is referred to as exponential or logarithmic growth. The primary parameter characterizing the growth rate is the specific growth rate. In many cases, all other growth parameters can be expressed in terms of the specific growth rate.

5.2.2. Biomass Doubling Time

The relationship between the specific growth rate and the biomass doubling time (td) can be derived by substituting into equation (4)

х = 2 х₀ и t = td,

then

td = l_n2 : μ = 0,693 : μ (7)

5.2.3. Multiplication Factor

The multiplication factor is defined by the ratio x/x₀, which equals e^μt. If the biomass undergoes n doublings or generations, the following expression can be written:

х/х₀ = 2ⁿ (8)

Thus,

n = 3,32 log(x/x₀) (9)

5.2.4. Economic Coefficient

If the growth of a microbial population is limited by a specific substrate, a linear relationship exists between the initial concentration of the limiting substrate added to the medium and the total yield obtained. Therefore, the mass of cells produced per unit of the limiting substrate represents a constant—the economic coefficient, or biomass yield (Y). The value of Y can be determined from the equation:

∆х/∆у = Y, (10)

where ∆x is the increase in biomass corresponding to the consumption of substrate in the amount of ∆y. The economic coefficient is determined by the limit approached by the ratio ∆x/∆y as ∆s —> 0, that is

Y = dх/ ds (11)

If x and s are the concentrations of biomass and substrate, respectively, then more strictly:

Y = - dх/ ds.

The minus sign is introduced because The values of x and s change in opposite directions.

If the external conditions in a bacterial culture are maintained constant, the economic coefficient will also remain a constant, quantitatively reproducible value. Thus, if x₀ and s₀ are the initial concentrations of biomass and substrate, respectively, and x and s are the corresponding concentrations during growth in the culture, then

х - х₀ = Y (s₀ - s) (12)

hence

Y = (х - Х₀) / (s₀ - s) (13)

Measured on The basis of the consumed organic substrate, the economic coefficient serves as an indicator of the efficiency of substrate conversion into bacterial biomass.

5.2.5. Metabolic Quotient

The rate of substrate consumption by a culture at a given time is expressed by the ratio:

ds/dt = qх, (14)

where x is the biomass, and the coefficient q is known as the metabolic quotient or specific metabolic rate. The metabolic quotient is analogous to enzymatic activity. If the biomass composition and the environment remain constant, then q should also be a constant value.

For substrate consumption over a short time interval dt, one can write

ds = (μ х : Y) dt, (15)

whence

ds/dt = μ х/Y (16)

Comparing equations (16) and (14), it becomes evident that

q =μ /Y (17)

Equation (17) is used to determine substrate requirements, particularly for oxygen, at various growth rates.

5.2.6. Biomass Yield

The biomass yield is defined as the difference between the maximum and initial bacterial mass: Х = Х_макс - Х₀. This value is expressed in grams of dry weight.