FUNDAMENTALS OF MICROBIOLOGY

Lesson 15. MODES OF CELL DIVISION

Module 5. Microbial growth and nutrition

Lesson 15

MODES OF CELL DIVISION

15.1 Binary Cell Division

Prokaryotes are much simpler in their organization than are eukaryotes. There are a great many more organelles in eukaryotes, also more chromosomes. The usual method of prokaryote cell division is termed binary fission. The prokaryotic chromosome is a single DNA molecule that first replicates, then attaches each copy to a different part of the cell membrane. When the cell begins to pull apart, the replicate and original chromosomes are separated. Following cell splitting (cytokinesis), there are then two cells of identical genetic composition (except for the rare chance of a spontaneous mutation). The prokaryote chromosome is much easier to manipulate than the eukaryotic one. We thus know much more about the location of genes and their control in prokaryotes. One consequence of this asexual method of reproduction is that all organisms in a colony are genetic equals. When treating a bacterial disease, a drug that kills one bacteria (of a specific type) will also kill all other members of that clone (colony) it comes in contact with. Binary fission begins with DNA replication. DNA replication starts from an origin of replication, which opens up into a replication bubble (note: prokaryotic DNA replication usually has only 1 origin of replication, whereas eukaryotes have multiple origins of replication). The replication bubble separates the DNA double strand, each strand acts as template for synthesis of a daughter strand by semi conservative replication, until the entire prokaryotic DNA is duplicated. (Fig. 15.1)

Fig. 15.1 Binary cell division in bacterial cell

After this replication process, cell growth occurs. Each circular DNA strand then attaches to the cell membrane. The cell elongates, causing the two chromosomes to separate.

Cell division in bacteria is controlled by the FtsZ, a collection of about a dozen proteins that collect around the site of division. There, they direct assembly of the division septum. The cell wall and plasma membrane starts growing transversely from near the middle of the dividing cell. This separates the parent cell into two nearly equal daughter cells, each having a nuclear body. The cell membrane then invaginates (grows inwards) and splits the cell into two daughter cells, separated by a newly grown cell plate. (Fig. 15.2)

Fig. 15.2 Binary fission in bacteria

15.2 Microbial Growth

When bacteria are inoculated into a liquid growth medium, we can plot of the number of cells in the population over time. (Fig. 15.3)

15.2.1 Four phases of bacterial growth

15.2.1.1 Lag phase

Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. This is the period of adjustment to new conditions. Little or no cell division occurs, population size doesn’t increase. This is the phase of intense metabolic activity, in which individual organisms grow in size. It may last from one hour to several days.

15.2.1.2 Log phase

Cells begin to divide and generation time reaches a constant minimum. This is the period of most rapid growth. The numbers of cells produced are more than the number of cells dying. Cells are at highest metabolic activity. Cells are most susceptible to adverse environmental factors such as antibiotic and radiation at this stage. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time (below) derive.

15.2.1.3 Stationary phase

By this stage, population size begins to stabilize. Number of cells produced is equal to number of cells dying. Overall cell number does not increase. Cell division begins to slow down. Factors that slow down microbial growth:

• Exhaustion of available nutrients;
• Accumulation of toxic waste materials
• Exhaustion of biological space
• Acidic pH of media
• Insufficient oxygen supply
• Cell functions necessary for growth will cease, but different functions necessary for survival are turned on.

The length of this phase is dependent upon the particular microorganism and the conditions of the medium (habitat) Stationary phase can last for long periods of time and especially when the microbes in nutrient-poor environment. During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process.

15.2.1.4 Death or decline phase

If incubation prolongs beyond stationary phase, a death phase ensues, in which the viable cell population begins to decline. Population size begins to decrease. This is the stage where number of cells dying starts exceeding number of cells produced. During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.

Fig. 15.3 Bacterial growth curve

15.3 Growth Rate and Generation Time

As mentioned above, bacterial growth rates during the phase of exponential growth, under standard nutritional conditions (culture medium, temperature, pH, etc.), define the bacterium's generation time. Generation times for bacteria vary from about 12 min to 24 h or more. The generation time for E. coli in the laboratory is 15-20 min, but in the intestinal tract, the coliform's generation time is estimated to be 12-24 h. For most known bacteria that can be cultured, generation times range from about 15 min to 1 h. Symbionts such as Rhizobium tend to have longer generation times. Many lithotrophs, such as the nitrifying bacteria, also have long generation times. Some bacteria that are pathogens, such as Mycobacterium tuberculosis and Treponema pallidum, have especially long generation times, and this is thought to be an advantage in their virulence. Generation times for a few bacteria are shown in Table (15.1)

Table 15.1 Generation times for some common bacteria under optimal conditions of growth

15.3.1 Calculation of generation time

When growing exponentially by binary fission, the increase in a bacterial population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide.

G (generation time) = (time, in minutes or hours)/n(number of generations)
G = t/n
t = time interval in hours or minutes
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
n = number of generations (number of times the cell population doubles during the time interval)
b = B x 2n (This equation is an expression of growth by binary fission)

Solve for n:

logb = logB + nlog2

n = (logb - logB) / log 2

n = (logb - logB) / 3.301

n = 3.3 logb/B

G = t/n

Solve for G:

G = t / (3.3 log b / B)

Example: Taking example of Lactococcus lactis. Suppose in milk medium initial count of is 10,000 and final count is 10,000,000 and time interval is 4.3 h then what should be the generation time of L. lactis in milk.

G = t / (3.3 log b / B)
Milk
G = 260 min/ 3.3 log 10⁷/10⁴

G = 260 min / 3.3 × 3

G = 26.26 min

Lactose broth

G = 478 min / 3.3 log 10⁷/ 10⁴

G = 478 min / (3.3 × 3)

G = 47.95 min

Similarly in milk medium initial count of milk is 10,000 and final count is 10,000,000 and time interval is 7.9 h then what should be the generation time of L. lactis in lactose broth

15.4 Synchronous Growth

A synchronous or synchronized culture is a microbiological culture or a cell culture that contains cells that are all in the same growth stage. Studying the growth of bacterial populations in batch or continuous cultures does not permit any conclusions about the growth behavior of individual cells, because the distribution of cell size (and hence cell age) among the members of the population is completely random. Information about the growth behavior of individual bacteria can, however, be obtained by the study of synchronous cultures. Synchronized cultures must be composed of cells which are all at the same stage of the bacterial cell cycle. Measurements made on synchronized cultures are equivalent to measurements made on individual cells.

Since numerous factors influence the cell cycle, some of them stochastic (random), normal, non-synchronous cultures have cells in all stages of the cell cycle. Obtaining a culture with a unified cell-cycle stage is very useful for biological research. Since cells are too small for certain research techniques, a synchronous culture can be treated as a single cell; the number of cells in the culture can be easily estimated, and quantitative experimental results can simply be divided in the number of cells to obtain values that apply to a single cell. Synchronous cultures have been extensively used to address questions regarding cell cycle and growth, and the effects of various factors on these.

A number of clever techniques have been devised to obtain bacterial populations at the same stage in the cell cycle. Some techniques involve manipulation of environmental parameters which induces the population to start or stop growth at the same point in the cell cycle, while others are physical methods for selection of cells that have just completed the process of binary fission. Theoretically, the smallest cells in a bacterial population are those that have just completed the process of cell division. Synchronous cultures rapidly lose synchrony because not all cells in the population divide at exactly the same size, age or time. Synchronous cultures can be obtained in several ways

• External conditions can be changed, so as to arrest growth of all cells in the culture, and then changed again to resume growth. The newly growing cells are now all starting to grow at the same stage, and they are synchronized. For example, for photosynthetic cells light can be eliminated for several hours and then re-introduced. Another method is to eliminate an essential nutrient from the growth medium and later to re-introduce it.
• Cell growth can also be arrested using chemical growth inhibitors. After growth has completely stopped for all cells, the inhibitor can be easily removed from the culture and the cells then begin to grow synchronously. Nocodazole, for example, is often used in biological research for this purpose.
• Cells in different growth stages have different physical properties. Cells in a culture can thus be physically separated based on their density or size, for instance. This can be achieved using centrifugation (for density) or filtration (for size).
• In the Helmstetter-Cumming technique, a bacterial culture is filtered through a membrane. Most bacteria pass through, but some remain bound to the membrane. Fresh medium is then applied to the membrane and the bound bacteria start to grow. Newborn bacteria that detach from the membrane are now all at the same stage of growth; they are collected in a flask that now harbors a synchronous culture.

15.5 Continuous Culture

The cultures so far discussed for growth of bacterial populations are called batch cultures. Since the nutrients are not renewed, exponential growth is limited to a few generations. Bacterial cultures can be maintained in a state of exponential growth over long periods of time using a system of continuous culture, designed to relieve the conditions that stop exponential growth in batch cultures. Continuous culture, in a device called a chemostat, can be used to maintain a bacterial population at a constant density, a situation that is, in many ways, more similar to bacterial growth in natural environments.

In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth is initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the growth chamber is maintained at a constant level by some sort of overflow drain. Fresh medium is allowed to enter into the growth chamber at a rate that limits the growth of the bacteria. The bacteria grow (cells are formed) at the same rate that bacterial cells (and spent medium) are removed by the overflow. The rate of addition of the fresh medium determines the rate of growth because the fresh medium always contains a limiting amount of an essential nutrient. Thus, the chemostat relieves the insufficiency of nutrients, the accumulation of toxic substances, and the accumulation of excess cells in the culture, which are the parameters that initiate the stationary phase of the growth cycle. The bacterial culture can be grown and maintained at relatively constant conditions, depending on the flow rate of the nutrients.

A microbial population of can be maintained in the exponential growth phase and at a constant biomass concentration for extended periods. Continuous Culture It is an open system where fresh medium is added and spent medium and cells are removed. Open systems are more complex due to the need to aseptically add and remove medium.

Two common major types of continuous culture systems:

15.5.1.1 Chemostat

A chemostat (from Chemical environment is static) is a bioreactor to which fresh medium is continuously added, while culture liquid is continuously removed to keep the culture volume constant. By changing the rate with which medium is added to the bioreactor the growth rate of the microorganism can be easily controlled. One of the most important features of chemostat is that micro-organisms can be grown in a physiological steady state. In steady state, growth occurs at a constant rate and all culture parameters remain constant (culture volume, dissolved oxygen concentration, nutrient and product concentrations, pH, cell density, etc.). In addition environmental conditions can be controlled by the experimenter. Micro-organisms grown in chemostat naturally strive to steady state: if a low amount of cells are present in the bioreactor, the cells can grow at growth rates higher than the dilution rate, as growth isn't limited by the addition of the limiting nutrient. The limiting nutrient is a nutrient essential for growth, present in the media at a limiting concentration (all other nutrients are usually supplied in surplus). However, if the cell concentration becomes too high, the amount of cells that are removed from the reactor cannot be replenished by growth as the addition of the limiting nutrient is insufficient. This results in an equilibrium situation (steady state), where the rate of cell growth is equal to the rate of cell removal.

Fig. 15.4 Chemostat continuous culture system

15.1.1.2 Turbidostat

A turbidostat is a continuous culture device, similar to a chemostat or an auxostat, which has feedback between the turbidity of the culture vessel and the dilution rate. The theoretical relationship between growth in a chemostat and growth in a turbidostat is somewhat complex, in part because it is similar. A chemostat technically has a fixed volume and flow rate - thus a fixed dilution rate. When the cells are uniform and at equilibrium, operation of a chemostat and turbidostat should be identical. It is only when classical chemostat assumptions are violated (for instance, out of equilibrium; or the cells are mutating) that a turbidostat is functionally different. One case may be while cells are growing at their maximum growth rate, in which case it is difficult to set a chemostat to the appropriate constant dilution rate.

While most turbidostats use a spectrophotometer/turbidometer to measure the optical density for control purposes, there exist other options, such as dielectric permittivity.

Fig. 15.5 Turbidostat continuous culture system

15.6 Diauxic growth

Diauxie is a French word coined by Jacques Monod to mean two growth phases. The word is used to describe the growth phases of a microorganism in batch culture as it metabolizes a mixture of two sugars. Rather than metabolizing the two available sugars simultaneously, microbial cells commonly consume them in a sequential pattern, resulting in two separate growth phases. During the first phase, cells preferentially metabolize the sugar on which it can grow faster (often glucose but not always). Only after the first sugar has been exhausted do the cells switch to the second. At the time of the "diauxic shift", there is often a lag period during which cells produce the enzymes needed to metabolize the second sugar.

Fig 15.6 The diauxic growth curve of E. coli grown in limiting concentrations of a mixture of glucose and lactose