General characteristics of microorganisms including their growth and reproduction, microbial techniques. • Economic importance of microorganisms.
BACTERIAL GROWTH
Bacterial growth is the asexual reproduction, or cell division, of a bacterium into two daughter cells, in a process called binary fission. The resulting daughter cells are genetically identical to the original cell. Hence, bacterial growth occurs. The measurement of an exponential bacterial growth curve in batch culture requires bacterial enumeration (cell counting) by different methods
In the laboratory, under favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23………2n (where n = the number of generations). This is called exponential growth. In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature.
When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve
Bacterial growth curve
Bacterial growth in culture occurs in four different phases:
Lag phase (A),
Log phase or exponential phase (B),
Stationary phase (C), and
Death phase (D)
During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs. During the lag phase cells change very little because the cells do not immediately reproduce in a new medium. This period of little to no cell division is called the lag phase and can last for 1 hour to several days. During this phase cells are not dormant.
The log phase (sometimes called the logarithmic phase or the exponential phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. For this type of exponential growth, plotting the natural logarithm of cell number against time produces a straight line. The slope of this line is the specific growth rate of the organism, which is a measure of the number of divisions per cell per unit time. The actual rate of this growth (i.e. the slope of the line in the figure) depends upon the growth conditions, which affect the frequency of cell division events and the probability of both daughter cells surviving. Under controlled conditions, Exponential growth cannot continue indefinitely, because the medium is soon depleted of nutrients and enriched with wastes.
The stationary phase is often due to a growth-limiting factor such as the depletion of an essential nutrient, and/or the formation of an inhibitory product such as an organic acid. Stationary phase results from a situation in which growth rate and death rate are equal. The number of new cells created is limited by the growth factor and as a result the rate of cell growth matches the rate of cell death. The result is a “smooth,” horizontal linear part of the curve during the stationary phase. Mutations can occur during stationary phase.
At death phase (decline phase), bacteria die. This could be caused by lack of nutrients, environmental temperature above or below the tolerance band for the species, or other injurious conditions.
This basic batch culture growth model draws out and emphasizes aspects of bacterial growth which may differ from the growth of macrofauna. It emphasizes clonality, asexual binary division, the short development time relative to replication itself, the seemingly low death rate, the need to move from a dormant state to a reproductive state or to condition the media, and finally, the tendency of lab adapted strains to exhaust their nutrients. In reality, even in batch culture, the four phases are not well defined. Near the end of the logarithmic phase of a batch culture, competence for natural genetic transformation may be induced, as in Bacillus subtilis and in other bacteria. Natural genetic transformation is a form of DNA transfer that appears to be an adaptation for repairing DNA damages.
Batch culture is the most common laboratory growth method in which bacterial growth is studied, but it is only one of many. It is ideally spatially unstructured and temporally structured. The bacterial culture is incubated in a closed vessel with a single batch of medium. In some experimental regimes, some of the bacterial culture is periodically removed and added to fresh sterile medium. In the extreme case, this leads to the continual renewal of the nutrients. This is a chemostat, also known as continuous culture. It is ideally spatially unstructured and temporally unstructured, in a steady state defined by the rates of nutrient supply and bacterial growth. In comparison to batch culture, bacteria are maintained in exponential growth phase, and the growth rate of the bacteria is known.
Bacterial growth can be suppressed with bacteriostats, without necessarily killing the bacteria. In a synecological, that is, true-to-nature situation in which more than one bacterial species is present, the growth of microbes is more dynamic and continual.
Environmental factors influence rate of bacterial growth such as acidity (pH), temperature, water activity, macro and micro nutrients, oxygen levels, and toxins. Conditions tend to be relatively consistent between bacteria with the exception of extremophiles. Bacteria have optimal growth conditions under which they thrive, but once outside of those conditions the stress can result in either reduced or stalled growth, dormancy (such as formation spores), or death. Maintaining sub-optimal growth conditions is a key principle to food preservation.
Temperature]
Low temperatures tend to reduce growth rate. Depending on temperature, bacteria can be classified as:
Psychrophiles
Psychrophiles are extremophilic cold-loving bacteria or archaea with an optimal temperature for growth at about 15 °C or lower (maximal temperature for growth at 20 °C, minimal temperature for growth at 0 °C or lower). Psychrophiles are typically found in Earth’s extremely cold ecosystems, such as polar ice-cap regions, permafrost, polar surface, and deep oceans.
Mesophiles
Mesophiles are bacteria that thrive at moderate temperatures, growing best between 20° and 45 °C. These temperatures align with the natural body temperatures of humans, which is why many human pathogens are mesophiles.
Thermophiles
Survive under temperatures of 45° – 60 °C
Acidity
Optimal acidity for bacteria tends to be around pH 6.5 to 7.0 with the exception of acidophiles. Some bacteria can change the pH such as by excreting acid resulting in sub-optimal conditions.
Other factors include
Water activity
Oxygen
Micronutrients
Toxins
Toxins such as ethanol can hinder or kill bacterial growth. This is used beneficially for disinfection and in food preservation.
Growth Rate and Generation Time
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 minutes to 24 hours or more. The generation time for E. coli in the laboratory is 15-20 minutes, but in the intestinal tract, the coliform’s generation time is estimated to be 12-24 hours. For most known bacteria that can be cultured, generation times range from about 15 minutes to 1 hour. 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.
Table 2. Generation times for some common bacteria under optimal conditions of growth.
| Bacterium | Medium | Generation Time (minutes) |
| Escherichia coli | Glucose-salts | 17 |
| Bacillus megaterium | Sucrose-salts | 25 |
| Streptococcus lactis | Milk | 26 |
| Streptococcus lactis | Lactose broth | 48 |
| Staphylococcus aureus | Heart infusion broth | 27-30 |
| Lactobacillus acidophilus | Milk | 66-87 |
| Rhizobium japonicum | Mannitol-salts-yeast extract | 344-461 |
| Mycobacterium tuberculosis | Synthetic | 792-932 |
| Treponema pallidum | Rabbit testes | 1980 |
Economic uses and benefits of microorganisms
Pharmaceutical industry: microorganisms are used in the production of Antibiotics,
vitamins, enzymes and organic acids
Food industry: used in the production of Dairy Products, bread making, food yeast; Lactic acid
bacteria (from the genus Lactobacillus) are essential for making yoghurt and cheese.
Molds are used in the fermentation of certain cheeses, especially blue cheeses like
Roquefort and Stilton. Baker’s yeast is a mainstay in the bakery.
Brewing industry: used in the production of Alcoholic Beverages
Microorganisms have been used as tools for the production of products for millennia. Even in ancient times, the ability to produce vinegar by allowing water to percolate through wood shavings was known and widely practiced. Likewise, the transformation of a yeast suspension into beer or a suspension of crushed grapes into wine was common knowledge.
These economic uses of microorganisms are the earliest examples of biotechnology. As the knowledge of bacteria and yeast-chemical behaviors grew, other biotechnological uses for the microbes were found. A few examples include the use of the bacterium Lactobacillus acidophilus to produce yogurt, the exploitation of a number of different bacteria to produce a variety of cheeses, and the fermentation of cabbage to produce sauerkraut. In the agricultural sector, the discovery of the ability of Rhizobium spp. to convert elemental nitrogen to a form that was useable by a growing plant, led to the use of the microorganism as a living fertilizer that grew in association with the plant species.
In more modern times, the use of microorganisms as biotechnological agents of profit has not only continued but has explosively increased.
The unraveling of the structure of DNA (deoxyribonucleic acid), various species of ribonucleic acid (RNA ), and the various processes whereby the manufacture of protein from the nucleic acid templates occurs was pivotal in advancing the use of microorganisms as factories. As important was the discovery of how to remove DNA from one region of the genome and move the DNA in a controlled way to another region of the same DNA, or DNA in a completely different organism (prokaryotic or eukaryotic). These gene-splicing technologies, which can be accomplished by various splicing and reannealing enzymes, or by the use of viruses or mobile regions of viral DNA (such as transposons) as vectors have allowed biotechnologists to create what are termed “designer genes,” which are designed for a specific purpose. This ability has fueled the use of microorganisms for economic gain and/or benefit.
The gene for the production of human insulin has been transferred into the genome of the common intestinal tract bacterium Escherichia coli . Successful expression and excretion of human insulin by the bacteria allows the production of a large amount of insulin. Additionally, because the insulin is identical to that produced in a human being, the chance of immune reaction against the protein is virtually nonexistent. The example of insulin reflects both the health benefit of the use of microbes and the economic benefit to be realized, since the mass production of insulin that is possible using bacteria lowers the cost of the product.
Other medical uses of microorganisms, particularly in the production of antibiotics, have been the greatest boon to humans and other animals. The list of maladies that can now be treated using microbiologically derived compounds is lengthy, and includes cystic fibrosis, hemophilia, hepatitis B, Karposi’s sarcoma, rejection of transplanted organs, growth hormone deficiency, and cancer.
Microorganisms have also been harnessed as factories to produce compounds that are used in areas as divers as textile manufacture, agriculture, and nutrition. Enzymes discovered in bacteria that can exist at very elevated temperatures (thermophilic, or “heat loving” bacteria) cab be used to age denim to produce a “pre-washed” look. Similar enzymes are being exploited in laundry detergent that operates in hot water. Microorganisms are used to enhance the nutritional content of plants and other food sources. The growing nutraceutical sector relies in part on the nutritional enhancements afforded by microbes. Bacteria are also useful in providing a degree of resistance to plants. An example is the use of Bacillus thuringensis to supply a protein that is lethal to insect when they consume it. The use of bacterial insecticides has reduced the use of chemical insecticides, which is both a cost savings to the producer and less stressful on the environment. Other bacterial enzymes and constituents of the organisms are utilized to produce materials such as plastic.
A process known as DNA fingerprinting, which relies upon enzymes that are produced and operate in bacteria, has enabled the tracing of the fate of genes in plant and animal populations, and enhanced gathering of evidence at crime scenes.
The mode of growth of bacterial populations has also proved to be exploitable as a production tool. A prime example is the surface-adherent mode of bacterial growth that is termed a biofilm. Although not known at the time, the production of vinegar hundreds of years ago was, as now, based on the percolation of water through biofilms growing on wood shavings. Immobilized bacteria can produce all manner of compounds. As well, the cells can provide a physical barrier to the flow of fluid. This dynamic aspect has been utilized in a so far small-scale way to increase the production of oil from fields oil thought to be depleted. Bacteria can plug up the zones were water and oil flows most easily. Subsequent pumping of water through the field forces the oil still resident in lower permeability areas to the surface.
With the passing of time, the realized and potential benefits of microorganisms and the implementation of strict standards of microbe use, is lessening the concern over the use of engineered microorganisms for economic and social benefit. The use of microorganisms can only increase.