Factors affecting the microbial growth

THE INFLUENCE OF ENVIRONMENTAL FACTORS ON GROWTH

Microbial growth is greatly affected by chemical and physical nature of their surroundings instead of variations in nutrient levels and particularly the nutrient limitation. For successful cultivation of microorganisms it is not only essential to supply proper and balanced nutrients but also it is necessary to maintain proper environmental conditions. Thus, understanding of environmental influences on the growth of microorganisms becomes mandatory. As bacteria shows divers food habits, it also exhibits diverse response to the environmental conditions. Growth and death rates of microorganisms are greatly influenced by number of environmental factors such as solutes and water acidity, temperature, oxygen requirement, pH, pressure and radiation.

Solutes and Water Acidity

Water is one of the most essential requirements for life. Thus, its availability becomes most important factor for the growth of microorganisms. The availability of water depends on two factors - the water content of the surrounding environment and the concentration of solutes (salts, sugars etc.) dissolved in the water.

In most cases, the cell cytoplasm possesses higher solute concentration in comparison to its environment. Thus, water always diffuses from a region of its higher concentration to a region of the lower concentration. This process is called osmosis which keeps the microbial cytoplasm in positive water balance. When a microbial cell is placed in hypertonic solution (or, solution of low water activity), it loses water and shrinkage of membrane takes place.

This phenomenon is called plasmolysis. Microorganisms show variability in their ability to adapt the habitats of low water activity. Microorganisms like S. aureus can survive over a wide range of water activity and are called as osmotolerant (as water activity is inversely related to osmotic pressure). However, most microorganisms grow well only near pure water activity (i.e. around 0.98-1). Thus, drying of food or addition of his concentration of salts and sugars is the most popular way of preventing spoilage of food. Seawa, microorganisms are called as halophiles since they require high concentration of salts (between 2.b 6.2 M) to grow. Halobacterium, a halophilic archaebacterium, inhabits Dead Sea (a salt lake situated between Israel and Jordan and the lowest lake in the world), the Great Salt Lake in Utah and other aquatic habitats possessing salt concentrations approaching salt water.

The quantitative availability of water can be expressed in physical term called water activity (aw). The water activity for a sample solution is the ratio of vapour pressure of the sample solution to the vapour pressure of the water at the same temperature (aw = Psoln/Pwater) The relative humidity of a test sample (at equilibrium) can be obtained, after sealing it in a closed chamber. This determines the water activity of a solution. For example, if after treating by above method relative humidity of air over the sample is 95% then, the water activity of the sample is 0.95

Temperature

All forms of life are greatly influenced by temperature. In fact, the microorganisms are very sensitive to the temperature since their temperature varies with that of environment (poikilothermic). Temperature influences the rate of chemical reactions and protein structure integrity thus affecting rates of enzymatic activity. At low temperature enzymes are not w denatured, therefore, every 10°C rise in temperature results in rise of metabolic activity and growth of microorganisms. However, enzymes have a range of thermal stability and beyond it their denaturation takes place. Thus, high temperature kills micro- CJ organism by denaturing enzymes, by inhibiting transport carrier molecules or by change in membrane integrity. Each microbe shows characteristic temperature dependence and possesses its own cardinal temperatures i. e. minimal, maximum and optimal growth temperatures.

The values for cardinal temperature vary widely among bacteria. For convenience, bacteria isolated from hot springs can survive even at temperature of 100°C and above, while those isolated from snow can survive below -10°C. On the basis of susceptibility to the thermal conditions, microorganisms are classified into three categories: thermophiles. Meshophiles and psychrophiles.

Thermophiles are microorganisms that show growth optima at 55°C. They often have growth maxima of 65°C, while few can grow even at 100°C and higher temperatures. Their growth minima is 45°C. The vast majority of thermophiles belongs to prokaryotes although a few microalgae (e.g., Cyanidium caldarium) and microfungi (e.g. Mucor pusillus) are also thermophiles. A few microorganisms are hyperthermophiles as they possess growth optima between 80°C and about 113°C. Hyper­thermophiles usually do not grow well below 55°C (e.g.. Pyrococcus abyssi, Pyrodictium occultum). Mesophiles are microorganisms that have growth minima between 15°C-20°C; optima between 20­45°C and maxima at 45°C. Most microorganisms fall within this category. Almost all human pathogens are mesophiles as they grow at a fairly constant temp. of 37°C. Psychrophiles have optimum temperature for growth at 15°C, however, few can grow even below 0°C. The maximum growth temperature of psychrophiles is around 20°C. The spoilage of refrigerated food takes place because of facultative psychrophiles. These are microorganisms that can grow at 0°C but have growth optima temperature between 20°- 30°C

Oxygen Requirements

The atmosphere of earth contains about 20% (v/v) of oxygen. Microorganisms capable of growing in the presence of atmospheric oxygen are called aerobes whereas those that grow in the absence of atmospheric oxygen are called as anaerobes. The micro-organisms that are completely dependent on atmospheric oxygen for growth are called obligate aerobes whereas those that do not require oxygen for growth but grow well in its presence are called as facultative anaerobes. Aerotolerants (e.g. Enterococcus faecalis) ignore O₂ and can grow in its presence or absence. In contrast, obligate anaerobes (e.g., Bacteroids, Clostridium pastewianum, Furobacterium) do not tolerate the presence of oxygen at all and ultimately die. Few microorganisms (e.g., Campylobacter) require oxygen at very low level (2-10%) of concentration and are called as microaerophiles. The latter are damaged by the normal atmospheric level of oxygen (20%)

Relationship of Oxygen and growth (Shake tubes method)

1. Aerobic

2. Microaerophilic

3. Facultative

4.Anaerobic

These varying relationships between microbes (especially the bacteria) and O₂ appear due to different factors such as protein-inactivation and the effect of toxic oxygen-derivatives. Bacterial enzymes can be inactivated when interact with oxygen. Nitrogen-fixing enzyme introgenese is very sensitive to oxygen and represents a good example of interaction between enzyme and oxygen. During metabolic process, flavoprotein reduces oxygen to form hydrogen peroxide (H₂O₂), superoxide radical (O₂^-) and hydroxy radical (OH^-). These compounds are highly toxic and being powerful oxidizing agent, can cause destruction of cellular macromolecules.

O₂ + e^- = O₂^- (superoxide radical)
O₂^- + e^- + 2H⁺ = H₂O₂ (hydrogen peroxide)
H₂O₂ + e^- + H+ = H₂O + OH^- (hydroxy radical)

In order to survive, therefore, bacteria must be able to protect itself from oxidising agents. All aerobes and facultative anaerobes contain two enzymes namely superoxide dismutase and catalase. These enzymes protect microbes against lethal effects of oxygen products. The oxidizing property hydrogen peroxide. The enzyme catalase decomposes hydrogen peroxide into oxygen and water. The aerotolerant bacteria like lactic acid bacteria possess enzyme peroxides instead of catalase to decompose the accumulated hydrogen peroxide.

2O₂^- 2H⁺ -----Superoxidase dismutase-----O₂ +H₂O₂
2H₂O₂ -----Catalase------ 2H₂O +O₂
H₂O₂ +NADH + H⁺ ----Peroxidase---- 2H₂O + NAD⁺

Since all obligate anaerobes lack these enzymes or have them in very low concentration and, therefore, are susceptible to oxygen.

Pressure

Normal life of microorganisms on land or on the water surface is always subjected to a pressure of 1 atmosphere. But, they are many microbes that survive in extremes of hydrostatic pressure in deep sea. Others are there that not only survive rather grow more rapidly at high pressures (e.g., Protobacterium, Colwellia, Shewanella) and are called barophilic. Some archaebacteria are thermobarophiles (e.g., Pyrococcus spp., Methanococcus jannaschii). However, A barophile has been recovered from the depth about 10,500 m in sea near Philippines and has been found unable to grow at 2°C temperature and below about 400-500 atmospheric pressure

Radiation

Some electromagnetic radiations, particularly the ionizing radiation (e.g., X-rays, gamma rays) are very harmful to microbial growth. Low levels of these radiations may cause mutations and may indirectly result in death whereas high levels may directly cause death of the microbes. Ionizing radiation, however, destroys ring-structures, breaks hydrogen bonds, oxidizes double bonds and polymerizes certain molecules. Ultraviolet (UV) radiation is lethal to all categories of microbial life due to its short wavelength and high-energy; the most lethal UV radiation has a wavelength of 260 nm. Ultraviolet radiation primarily forms thymine dimers in DNA to cause damage. Two adjacent thymines in a DNA strand join each other covalently and inhibit DNA replication and function. Microbial photosynthetic pigments (chlorophyll. bacteriochlorophyll, cytochromes and flavins), sometimes, absorb light energy, become excited or activated, and act as photosensitizers. The excited photosensitizer (P) transfers its energy to oxygen which then results in singlet oxygen (¹O₂), The latter is very reactive and powerful oxidizing agent and quickly destroys a cell. The singlet oxygen is probably the main weapon employed by phagocytes to destroy engulfed bacteria.

Microbial (Bacterial) Growth

MICROBIAL GROWTH

Growth can be defined as an orderly increase of all chemical compounds in a cell/population resulting in an increase in mass of the cell/population, i.e. the increase in mass is always accompanied by a comparable increase in all other measurable properties of the cell/population during growth. Therefore, an increase in mass might not really reflect growth until it is accompanied by an orderly increase in all chemical components because the cell/population can simply take up water or increase storage materials, thereby increasing only size and weight. Growth is followed by cell-division resulting in cell number. Under ordinary conditions of growth, all actively growing cells multiply by asexual process of cell division and this process can continue indefinitely, provided food and energy are available, and environmental conditions remain favorable. In multicellular organisms, an increase in the size of an individual is the indication or growth, whereas for unicellular organisms, it leads to an increase in the number of individuals. When a baby elephant grows, it increases in size and not in number although there is continuous increase in the cell number inside the body of the baby elephant. But, when a bacterium grows, it ultimately increases in number, but their size remains relatively unchanged. Although most of the cellular microorganisms exhibit essentially similar growth characteristics in vitro, we will briefly discuss the growth characteristics of bacteria in the following sections.

Generations Time of Bacteria

Most bacteria reproduce by binary fission, which results in doubling of the number of viable bacterial cells. Therefore, during active bacterial growth, the number of bacterial cells and, hence their population, continuously doubles at specific time intervals because each binary fission takes a specific duration of time. This specific time interval between two subsequent binary fissions is known as generation time or doubling time.

If we start with a single bacterial cell, its fission proceeds as a geometric progression (exponential growth) with one cell , dividing to form two, these two to four, further to eight and so on i.e. each succeeding fission (generation), assuming no cell death, doubles the population size

1 = 2¹ = 2² = 2³ = 2⁴ = 2⁵ =.........2ⁿ

Geometric progression (n) = the number of generations

Generation time or doubling time varies considerably among different bacteria (Table 8.6). A bacterium such as E. coli enjoys generation time as short as 20 minutes under optimal conditions, although in nature many bacteria have generation times of several hours. One cell of E. coli with a 20 minute generation time, for convenience, will multiply to 512 cells in 3 hours, to 4096 cells in 4 hours, and to 32768 cells in 5 hours, and so on.

Generation time for some Bacteria under Optimum Conditions

Bacterium	Medium	Temp(0C)	Generation time (min.)
Bacillus thermophilus	Broth	55	18.3
Escherichia coli	Broth	37	20
Bacillus subtilis	Broth	37	27
Streptococcus lactis	Milk	37	30
Lactobacillus acidophilus	Milk	37	66-87
Mycobacterium tuberculosis	Synthetic	37	792-932
Treponema pallidum	Rabbit testes	37	1980

Mathematical Expression of Growth
To calculate the generation time of individual microorganisms the following experimental data are required:
1. The number of organisms present at the beginning.
2. The number of organisms present at the end of a given time interval.
3. The time interval.
The relationship of cell numbers and generations can be expressed in a series of equations. Starting with a single cell, the total population N at the end of a given time period would be expressed as
N = 1 x 2ⁿ
where 2n is the bacterial population after n generations. However, under practical conditions several thousands of bacteria are introduced into the medium at zero time and not one, so the formula now becomes.

N = N_o x 2ⁿ

Where N_o = number of organisms at zero time.

N = number of organisms after n generations.

n = number of generations.

Solving the equation for n, we have

log N = log N_o + n log 2

n= (log N-log N_o) / log2

Thus we can calculate the number of generations if we know the initial population N_o and the population N after time t. The generation time G is equal to t (the time which elapsed between N_o and N) divided by the number of generations n, or

G = t/n = t log2 / (log N-log N_o)

An alternative method is used to describe bacterial growth in mathematical terms when the culture is undergoing balanced growth. The rate of increase in bacteria at any particular time is proportional to the number or mass or bacteria present at that time.

Relationship of generation time and growth.

The constant of proportionality is an index of the rate of growth and is called the exponential growth rate constant (K). It is defined as number of doublings in unit time, and is usually expressed as the number of doubling in an hour. It is calculated from the following equation:

N = Population at time t.

N_o = Population at time zero.

Taking the logarithms

log N = log N_o + Kt log 2, and

Solving the equation for K

K = (log N-log N_o) / t log2

The exponential growth rate constant is therefore reciprocal to generation time, i.e.

G=1/K
For example, generation time of E. coli is 20 minutes, i.e. t hour.

1/3=1/K
K = 3 doublings per hour.

Normal Growth Cycle (Growth Curve) of Bacteria:

Batch-Culture When a bacterium is inoculated into a flask containing fresh culture medium and incubated, it enters a rapid growth phase during which the bacterium divides by subsequent binary fissions and increases its population in the flask medium. Since the bacteria are not transferred to a new medium or no fresh nutrients are added to the medium, the increasing population a: of bacterial cells, after sometime, enters into a stationary-phase with the exhaustion of the required nutrients and the accumulation of inhibitory end products in the medium. Eventually, the stationary phase of bacterial population culminates into death-phase when the viable bacterial cells begin to die.

If we collect data on the increase in cell number at various intervals of time and plot this data in two ways (logarithm of number of bacteria and arithmetic number of bacteria versus time), we find a characteristic growth curve. This typical growth curve is obtained only in a batch culture, a culture in which the medium is retained in a limited volume and is not supplied with fresh nutrients at any stage of growth, The characteristic growth curve obtained in a batch culture has four phases: the lag phase, the log or exponential growth phase, the stationary phase, and the death or decline phase.

Characteristic Growth curve showing lag, log, stationary and death phase

Lag Phase

Lag phase represents a period of active growth during which bacteria prepare for reproduction, synthesizing DNA, various inducible enzymes, and other macromolecules needed for ceIl division. Therefore, during this phase, there may be increase in size (volume) but no increase in cell number. The lag phase may last for an hour or more, and near the end of this phase some cells may double or triple in size

The lag phase is necessary before the initiation of cell division due to variety of reasons. If the cells are taken from an old culture or from a refrigerated culture, it might be possible that the cells may be old and depleted of ATP, essential cofactors and ribosomes. If the medium is different from the one in which the microbial population was growing previously, new enzymes would be needed by the cells to use new nutrients in the medium. However, these deficiencies are fulfilled by the cells during lag phase. It is, therefore, the lag phase is generally longer if the cells are taken from an old or refrigerated culture. In contrast, if the cells are taken from young, vigorously growing culture (microbial population) and inoculated to a fresh medium of the identical composition, the lag phase may be short or even absent.

Log or Exponential Growth Phase

Bacterial cells prepared for cell division during lag phase now enter into the log phase or exponential growth phase during which the cells divide at a maximal rate and their generation time reaches a minimum and remains constant. The growth in this phase is quite balanced (i.e. all cellular constituents are synthesized at constant rates relative to each other) hence, the most uniform in terms of chemical and physiological properties, the log phase cultures are usually used in biochemical and physiological studies. Since the generation time is constant, a logarithmic plot of growth during log phase produces an almost a straight line. This phase is called log phase because the logarithm of the bacterial mass increases linearly with time, and exponential growth phase because the number of cells increases as an exponential function of 2ⁿ (i.e. 2¹, 2², 2³, 2⁴, 2⁵ and so on). The log phase also represents the time when bacterial cells are most active metabolically, and in industrial production, this is the period of peak activity and efficiency.

Stationary Phase

Since the bacteria are growing in a constant volume of medium of batch culture, and no fresh nutrients are added, the growth of bacterial population eventually ceases and the growth curve becomes horizontal. Such a phase of growth in bacteria is attained at a population level of around 109 cells per ml. The cessation of growth may be because of the exhaustion of available nutrients or by the accumulation of inhibitory end products of metabolism. The cessation of growth may also be due to O₂ availability particularly in case of aerobes. Oxygen is not very soluble and may be depleted so quickly that only cells on the surface of the culture may find necessary oxygen concentration for adequate growth.

Sooner or later, the bacterial cells start dying and the number of such cells balances the number of new born cells, and the bacterial population stabilizes. This state of growth, during which the total number of viable cells remains constant because of no further net increase in cell number and the growth rate is exactly equal to the death rate, is called stationary phase.

The transition between the log or exponential and stationary phases involves a period of unbalanced growth during which the various cellular components are synthesized at unequal rates. Consequently, cells in the stationary phase have a different chemical composition from those in the exponential phase.

Death or Decline Phase

After a while, the number of dying cells begins to exceed the number of new-born cells and thus the number of viable bacterial cells present in a batch culture starts declining. This condition represents the death or decline phase which continues until the population is diminished to a tiny fraction of more resistant cells, or it may die out entirely. Like exponential growth, death is also exponential, but inverse, as the number of viable bacterial cells decreases exponentially.

What happens when a bacterial population enjoying the exponential growth phase in a batch ­culture is inoculated into a fresh medium?

The fate of the bacterial population transferred to a fresh medium from a batch-culture medium depends on two conditions: (I) the fresh medium is identical in composition to the original medium, or (II) it differs significantly from the original medium in composition. If the medium is of same composition, the lag phase is usually bypassed by bacteria and exponential growth continues because bacteria are already actively carrying out the metabolic activities necessary for continued growth. But if the fresh medium differs significantly from the original one in chemical composition, the bacteria enter first into the lag phase to synthesize the enzymes necessary for exponential growth in the new medium and then enter the exponential growth phase.

CONTINUOUS GROWTH: CONTINUOUS CULTURE

Contrary to the studies in batch culture where the exponential growth of bacterial population is restricted only for a few generations, it is often desirable to maintain prolonged exponential growth of bacterial population for genetical and biochemical studies, and in industrial processes. This condition is obtained by growing bacteria in a continuous culture, a culture in which nutrients are supplied and end products continuously removed.

A continuous culture, therefore, is that in which the exponential growth phase of bacterial population can be maintained at a constant rate (steady state growth) for over a long period of time by continuously supplying fresh medium from a reservoir to, growth chamber and continuously removing excess volume of culture medium of growth chamber through a siphon overflow. By doing so the microbes never reach stationary phase because the end products do not accumulate to work as inhibitory to growth and nutrients are not completely expended.

Chemostat and Turbidostat

Continuous culture systems can be operated as chemostats or as turbidostats. In a chemostat the flow rate is set at a particular value with the help of a flow rate regulator and the rate of growth of the culture adjusts to this flow rate. That is, the sterile medium is fed-into the vessel at the same rate as the media containing microorganisms is removed.

Chemostat, a continuous culture system

1. Reservoir of Sterile Medium (Fresh)	5. Passage for Inoculation and Air Outlet
2. Flow Rate Regulator	6. Siphon Overflow
3. Air inlet	7. Growth Chamber
4. Air Filter	8. Receptacle

In a turbidostat the system includes an optical sensing device (photoelectric device) which continuously monitors the culture density in the growth vessel and controls the dilution rate to maintain the culture density at a constant rate. If the culture density becomes too high the dilution rate is increased, and if it becomes too low the dilution rate is decreased.

The turbidostat differs from the chemostat in many ways. The dilution rate in a turbidostat varies rather then remaining constant, and its culture medium lacks a limiting nutrient. The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at low dilution rates.

Turbidostat

1. Reservoir of Sterile Medium	4. Photo cell
2. Valve Controlling Flow of Medium	5. Light Source
3. Outlet for Spent Medium	6. Turbidostat

Synchronous Growth: Synchronous Culture

Synchronous growth of a bacterial population is that during which all bacterial cells of the population are physiologically identical and in the same stage of cell division cycle at a given time. Synchronous growth helps studying particular stages or the cell division cycle and their interrelations.

In most of the bacterial cultures the stages of growth and cell division cycle are completely random and thus it becomes difficult to understand the properties during the course of division cycle using such cultures. To overcome this problem, the microbiologists have developed synchronous culture techniques to find synchronous growth of bacterial population. Synchronous culture is that in which the growth is synchronous i.e., all the bacterial cells of the population are physiologically identical and in the same stage of cell division cycle at a given time

A synchronous culture can be obtained either by manipulating environmental conditions such as by repeatedly changing the temperature or by adding fresh nutrients to cultures as soon as they enter the stationary phase, or by physical separation of cells by centrifugation or filtration

An excellent and most widely used method to obtain synchronous cultures is the Helmstetter-Cummings Technique in which an unsynchronized bacterial culture is filtered through cellulose nitrate membrane filter.

Helmstetter Cumming technique of obtaining synchronous cultures

1. Culture

2. Filter

3. Fresh Medium

4. Baby Cells

The loosely bound bacterial cells are washed from the filter, leaving some cells tightly associated with the filter. The filter is now inverted and fresh medium is allowed to flow through it. New bacterial cells, which are produced by cell division and are not tightly associated with the filter, are washed into the effluent. Hence, all cells in the effluent are newly formed and are, therefore, at the same stage of growth and division cycle. The effluent thus represents a synchronous culture.

Growth Characteristics

Each bacterium when grows on a medium, shows specific growth appearance or characteristics. These specific characteristics are generally useful in the identification of unknown culture of bacteria. To observe growth characteristic, the bacterial culture is cultivated either on the surface of solid medium (agar plates and agar slope) or in liquid medium (broth).

To study the growth of bacteria on agar plate and agar slant following parameters are considered

Culture Characteristics of Bacteria

1. Punctiform

2. Granular

3. Circular

4. Rhizoid

5. Filamentous

6. Irregular

Growth on Agar Slant

1. Piliform

2. Echinulate

3. Beaded

4. Effuse

5. Arboriscent

6. Rhizoid

1. Size of the colony: small, large, or spreading.
2. Elevation, margin and type of colony on the surface.
3. Pigmentation: pigmented (red, brown, yellow and violet) or nonpigmented.
4. Optical features: opaque, translucent or opalescent.
The broth cultures can be examined to find the amount of growth, distribution of growth and order of growth. The growth can be observed on surface, all over or settled at the bottom.
Every bacterial type exhibits peculiar growth characteristics. Thus, analysis of culturing characteristics of bacteria becomes useful in their identification. However, one has to carry out additional tests such as biochemical, morphological and serological to identify the species.