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Growth

 

Introduction
Reproduction
The Growth curve of  Bacterial Population

Contineous Culture of Microorganisms

Synchronous Culture
Quantitive Measurement of Bacterial Growth
Enzymes
Variation

 

Introduction

Growth of an organism is defined as an orderly increase in the quantity of all of the cellular components or structures (mass).Increase of mass may not really reflect growth, because cells can simply take up water or deposit storage products such as lipid or glycogen, thereby increasing the size and weight of the cell. Growth is followed by cell division, resulting in an increase in cell number-. Under ordinary condi­tions of growth all actively growing cells (plants, animals and micro­organisms) multiply by the asexual process of cell fission. This does not include viruses, as they are not considered cellular. Fission results in division of the cell into two cells. This asexual reproductive process can continue indefinitely, provided food and energy are available, and environmental conditions remain favourable.

Among multicellular plants and animals, fission of body cells results only in increase in size of the individual plant or animal, not in increase in the number of individuals. The term growth applied to unice­llular microorganisms refers to the change in the entire crop of the organisms rather than a change in the individual organisms. Thus the organisms have not become bigger, but there are more organisms. In another sense growth means the same thing in multicellular organisms. For example, a baby elephant grows into an adult elephant not because each of its cells is larger, but because the adult has more cells. There­fore the term growth means the development of more cells. Microbial growth rate is very high. Microorganisms, especially bacteria, increase in number very rapidly under ideal conditions. It is believed that the rapid growth of bacterial cells is due to their large surface-to-volume ratio. In order to maintain their small size and this favourable ratio, cell division must also occur rapidly.

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Reproduction

All growing cells must divide. Most bacteria multiply by transverse, binary fission, in which a single cell divides in two after developing a transverse cell wall. Within a short period, often as short as 20 minutes, a bacterium can create a complete duplicate of itself, which in turn is capable of duplicating. The life cycle of a bacterium consists

of the time between one cell division and the next. Obviously, the degree of morphological changes within this life cycle is slight, when one compares it with that of a eukaryote that develops from a fertilized egg to a complex organism. However, complex biochemical events do occur, and the process requires the synthesis of the millions of parts. These parts are organized into the various homo and heteropolymers, which make up the sub-cellular organelles, and are the functional units of the cell.

As bacteria are extremely small, there are numerous technical difficulties in observing and interpreting the intricate cytological events that take place during the process of reproduction. However, with the help of special procedures for chemical analysis and new microscopic techniques, particularly electron microscope examination of ultra thin sections and time-lapse photography, the following developments are postulated.

1.Transfer of nutrients from the medium into the cell by a selective process.

2.Conversion of the nutrients by the enzyme system of the cell into protoplasmic material characteristic of the particular organism.

3. Increase in the amount of nuclear material.

4. Cell elongation (more evident in bacilli).

5. Organization of the contents of the cell to distribute the material between two cells.

6. Invagination of the cytoplasmic membrane, followed by the formation of a transverse cell wall.

7. Separation of daughter cells.

The daughter cells may stick together to form a pair, which after another cell division in the same plane would give rise to a chain of four cells. If the second cell division is at right angles to the plane of the first division, the result would be four cells in a flat configuration. Repeated divisions at right angles to each other result in planar sheets or "windowpanes" of cells, as produced by Lampromedia. When divi­sion occurs in a third plane, cubical packets of cells result, as produced by Sarcina.

1. Growth rate and generation time. The most common process of reproduction in bacteria is by binary fission, in which one cell divides to produce two cells. Thus the increase in population is by geometric progression :

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The time required for the cell to divide or for the population to double is known as the generation time. Not all microorganisms have the same generation time; for some it may be 15 minutes and for others it may be many hours, as shown in Table  Similarly, the generation time is not the same for a particular organism under all conditions. It is strongly dependent upon the nutrients in the medium and on environmental conditions of growth. Microorganisms are capable of growing over a wide range of physical conditions, and of utilizing many different nutrients, but maximum growth requires certain specific conditions for a given species.

Under optimal conditions the generation time determines the rate of growth of a microbial culture. The growth increment can be quantitatively, analysed by the determination of generation time.

Table. Generation times of certain species of bacteria

Bacterium

Medium

Temperature

Generation time (minutes)

Bacillus subtilis

Broth

37C

25

Ba. stearothermophulus

Broth

60C

8

Esclicricliia coli

Broth

37-C

20

-do-

Milk

37C

12

Staphylococcus aurcus

Broth

37C

30

Streptococcus lactis

Milk

37C

26

Mvcobacterium tuberculosis

Synthetic

37C

800 to 900

Nitrobacier agiiis

Synthetic

27C

1200

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The Growth Curve Of Bacterial Population

Bacterial population curves are determined by inoculating a small number of organisms into a culture medium and counting the bacteria in aliquot samples at regular intervals (e g. every hour) for 24 hours. A count made by microscopic examination represents the total number of cells. Dilution or plate counts indicate only the population of living cells. When the logarithms of the viable cells are plotted against time, a growth curve of the type illustrated in Fig. is obtained. From this it can be seen that there is an initial period of what appears to be no growth, followed by rapid growth, then leveling off, and finally a decline in the viable population. Between each of these phases there is a transitional period(curved portions). The growth curve is divided into eight phases:

Fig. Bacterial growth curve

Initial sationary phase                    

Phase of accelerated growth

Logarithmic growth phase          

Phase of decreasing growth rate 

Maximum stationary phase       

Phase of increasing death rate   

Logarithmic death phase             

Survival phase or phase decreasing death rate

1. Initial stationary phase. This phase is characterized by a period during which there is no increase in the number of cells. It is the phase of cell enlargement sometimes known as the lag phase- The latter name is somewhat misleading, because it implies inactivity or dormancy, which is contrary to the actual situation. Though there is no increase in the number of cells, the organisms are very active physiologically, and are synthesizing new protoplasm. There is an increase in total protein, ribonucleic acid, and cell phosphorus. It is a phase of adjustment necessary for the synthesis of the internal supply of intermediate metabolites, enzymes and coenzymes. Time is also required for adjustments in the physical environment around each cell. The duration of this phase varies with conditions and species. When a medium is inoculated with cells obtained from a culture growing in its logarithmic phase, the culture displays little lag in population increase. This phase is prolonged, however, in a medium inoculated with dormant cells from an older culture. In short, the organisms are metabolizing and growing, but there is a lag in cell division.

2. Phase of accelerated growth. After the end of first phase, each organism starts dividing. However, since not all organisms have comp­leted  the first   phase   simultaneously,   there is a gradual increase in the population  until the end of this period. On plotting, the rate of multiplication increases with time, i.e., the time required for each cell to divide gradually decreases. Finally, the rate of multiplication reaches a maximum at the end of this phase. This is the transition period between the initial stationary  phase  and logarithmic  growth phase. During this period the  ceils are unusually sensitive to unfavourable environmental conditions   such   as  extremes   of   temperature, high osmotic pressure, or disinfectant chemicals.

3. Logarithmic growth phase. During this phase the cells divide steadily at a constant rate, and the log of the number  of cells plotted against time results in a straight line.  In this phase the cells are in a state of balanced growth. During this state the mass and volume of the cells increase by the same factor,  in such a manner that the average composition  of cells and the relative concentration of metabolites remain constant  for a certain period of time. Cells are smaller in this phase because they are constantly dividing. It is a phase of physiologi­cal youth where cells  are actively growing and multiplying, and the entire population is uniform with respect to cellular activity. Biochemi­cal and physiological properties that are commonly used for identifica­tion of organisms are usually most manifest during this phase, Also, organisms are highly sensitive to various physical and chemical agents. In short this phase is characterized by the following :

(i)    Growth rate is maximum and constant.

(ii)   Generation time is shortest and constant.This phase lasts for several hours, depending upon the type of species and conditions of growth

4. Phase of decreasing growth rate. The organisms continue to multiply, but at a slower rate than during the logarithmic growth phase. The decline in rate of multiplication is due to a number of factors. The most important factors are the depletion of nutrients and the accumula­tion of toxic waste products. This is a transitionary phase between the logarithmic growth phase and the maximum stationary phase.

5. Maximum stationary phase. In this phase a constant high popu­lation of cells is maintained by a  balance between cell division and cell death. The net viable population remains unchanged for some time. The total count  (living plus dead)  continues to increase slowly, and can be used to calculate the rate of death.

6. Phase of increasing death rate. In this phase there is a decrease in number of viable organisms with increase in time. The rate of death gradually increases, and reaches a maximum at the end of the phase. This is a transitionary phase between the maximum stationary phase and the logarithmic death phase.

7. Logarithmic death phase. The number of organisms decreases exponentially during this phase, i.e. half the surviving cells die in each successive equal time interval. For example, a population decreases from 1 million to 1 /2 million in the first hour, to 1 /4 million in the next hour to 1/8 million in the third hour, and so forth. Thus the rate of death is constant, and (he rate can be calculated by the same formula used to calculate the rate of exponential growth. The result in this case is a minus quantity, and indicates the time required to diminish the population by 50 per cent. The same method can be used to compare the effectiveness of germicidal agents.

. A variety of conditions contribute to bacterial death, but the most important are depletion of nutrients and accumulation of toxic waste products. Bacteria die at different rates, just as they grow at different rates. Some species, e. g. Neisseria gonorrhoea, die very rapidly, This organism is particularly susceptible to autolysis, presumably the result of digestion by enzymes present in the bacterial cells. The steepness and duration of the death phase depends in part upon the nature and concentration of the toxic waste products.

8. Survival phase or phase of decreasing growth rate. In this phase the rate of death decreases, and finally an equilibrium is reached, such that both the rate of death and the rate of growth tend to balance each other at a very low level of population. Survival of the cell depends upon the types of organisms. Some die off with 3 to 4 days, while other types may remain viable for months and even years. The formation of the spore provides a resistant form of the cell that may survive long after all the vegetative ceils are dead. It often happens that mutant cells, which are present in small numbers, find enviornmental condi­tions more suitable for rapid growth than the parent types. Altered environmental conditions create selective conditions favouring the more rapid growth of certain mutant cells.

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Continuous Culture Of   Microorganisms

The typical bacterial growth  curve includes  three transitional periods between growth phases. This means that not all the cells are in   identical  physiological conditions.  Time is  required   for  some cells to catch up with others. In terms of physiological  conditions, the growth curve  includes young, actively metabolising cells on one hand, and the cells in the process of dying on the other, with cells in between these extremes. The effect of chemical substances and physical conditions on the organisms is not identical in all phases of growth. To study the  metabolism  of an organism for experimental research or industrial processes,  it is often desirable to maintain a microbial population in, the   logarithmic  phase   of   growth   in   a   constant   environment. This   is   accomplished, by a technique   called continuous   culture of microorganisms.

A variety of different pieces of apparatus has been developed to grow  microorganisms  in continuous culture.  The   chemostat is a

Fig. The Chemostat.

continuous system cultivator in which the medium is throughly mixed to obtain maximum homogeneity. The fresh nutrient medium flows into the culture vessel from a reservoir of sterile medium at a defined and constant rate. The volume in the culture vessel is kept constant by a device that allows the culture medium, together with accumulated waste products and older and dead cells, to leave the culture vessel at the same rate (Fig.)  The level of growth is controlled by maintaining a fixed, limiting concen­tration of a particular nutrient in the medium.

Fig.  The cell crop of a bacterial culture

The remai­ning constituents essential for growth of the selected orga­nism are added in the medium in excess of requirement. The growth-limiting nutrient is added into the medium at a concentration below that required for maximum growth in a batch culture, i.e., a closed vessel trates  the effect of a single Hinting nutrient on the final and total cell crop of a bacterial culture.

The turbidostat is another continuous culture apparatus. In a turbidostat the system includes an optical-sensing device which measures the absorbancy of the culture density(turbidity)in the growth vessel. Changes in turbidity retard (or increase) passage of light through the culture. These changes activate mechanisms that control the flow of nutrient into, and the flow of waste out of, the main culture vessel.

Chemostat and turbidostat are usually operated at different dilution rates. The dilution rate is the ratio of inflowing amount of nutrient medium per hour to the volume of the culture. In the chemostat, maximum stability is attained within a range of dilution rates over which cell density changes only slightly with changes in dilution rates, i. e. at low dilution rates. In contrast, in the turbidostat, maximum sensitivity and stability are achieved at high dilution rates, within a range over which culture density changes rapidly with dilution rate

Fig.   The Turbidostat

The dialysis technique is another device which maintains the culture in the logarithmic phase for a slightly longer period. In this device fresh nutrients are always available to the culture, and waste products are continually removed. However, the population pressure is the factor that ultimately limits growth

Continuous culture systems offer valuable advantages. They provide a constant source of cells in the logarithmic phase of growth for the study of physiology and genetics of the organisms. Secondly, these systems allow the cells to be grown continuously in limi­ting concentrations of the nutrient. Such growth gives valuable information on the catabolism of the limiting substrate. The system can be combined with selective enrichment to isolate an organism which can utilize any particular type of compound as a nutrient. This is very important in getting rid of a common indus­trial waste product or a poisonous pollutant.

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Synchronous Cultures

Synchronous cultures are composed of populations of cells that are at the same stage of their life cycle. All the cells in the culture will divide at the same time, will grow for a generation time, and all will divide again at the same time. Thus the entire population is kept uniform with respect to growth and division. It is not possible to analyse a single bacterial cell to obtain the information about growth behaviour, i.e., organisation, differentiation and macromolecular synthesis. Synchronous culture provides the entire cell crop in the same stage of growth. Measurement made on such cultures are equi­valent to the measurement made on individual cells.

Synchronous cultures of bacteria can be obtained by a number of techniques. Two fundamentally different experimental approaches have been employed. In the firs! approach, a synchronous population of cells can be sorted out according to age or size by physical separa­tion of cells. In methods of second type, a cultute is induced by manipulating the physical environment or the chemical composition of the medium to obtain a synchronously dividing population. The techniques based on selection are preferable to those based on induc­tion, since induction is likely to introduce distortions in the physiologic state of the cells.

1. Selection by size and age. A population of cells is fractio­nated on the basis of size. The cells are filtered so that smallest cells pass through the filter. These small cells are the youngest, and must go through their whole life cycle before dividing. Alternatively, the largest cells, which are ready to divide, may be retained or retarded by a filter. These are then collected separately and used to obtain a synchronous culture. The most widely used method for obtaining synchronous cultures is the Helmstetter-Cummings technique. A population of cells is passed through a membrane filter of pore size small enough to trap bacteria in the filter. The filter is then inverted, and fresh nutrient medium-is allowed to flow through it .After loosely associated bacteria are washed from the filter, the only bacterial cells in the effluent stream of the medium are those which arise through division. If a sample of this stream is collected over a short period of time, all the cells in this sample are newly formed, and are therefore of the same age and will divide synchronously.   The method has one disadvantage, that the population size is very small and changes in

Fig.   Helmstetter - Cummings filter pad technique

cellular constituents, therefore, cannot be measured. Instead of filtra­tion, density gradient centrifugation is also used to separate the cells. A population of unsynchronised cells is separated into fractions, each composed of the cells of the same density and at the same stage in their life cycle.

2. Selection by induction technique. A synchronous culture is also obtained by the use of shock treatments. These include variation in temperature, starvation, exposure to light (for photosynthetic organisms), drugs, and sub-lethal doses of radiation. A commonly used technique involves submitting a culture of microorganisms to single or multiple changes in temperature. An exponentially growing culture at 37C is held for about 30 minutes at 20C. The lower temperature retards cell division. During the interval of 30 minutes all the cells mature to the point of fission- However, at 20C none divide. On sudden return of the culture to 37C, all the cells divide synchronously. By repeating the alterations of temperature, synchrony can be maintained in the culture for several generations.

Methods  of inducing synchronous division based on changes in medium  composition,  have also   been   used.  Auxotrophic   microorganisms can be subjected to a period of starvation in a medium lacking an essential growth factor before being placed in a nutritionally complete medium. A phasing of cell division is observed in cultures of a thymine-requiring mutant, following withdrawal and readdition of thymine to the culture medium.

The time-course pattern of synthesis of various macromolecules in the cell cycle is studied by remo­ving portions of a synchronously dividing culture. The cells are then analysed for the content of macro-molecules or enzyme activity. The logarithmic plots show stepwise increase in cell members. However, the optical density of the culture increases exponentially, since opti­cal density measures cells mass, and mass is increasing. Similarly, the total synthesis of DNA, RNA, and protein increases exponen­tially

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Quantitative Measurement Of Bacterial Growth

The term growth as commonly employed in bacteriology refers to the magnitude of the total population. Growth can be determined by various techniques, based on one of the following types of measurement.

1.       Determination of the number of cells.

(a)  Directly, by microscopy or an electronic particle counter.

(b)  Indirectly, by a colony count.

2.       Determination of cell mass.

(a) Directly, by weighing or measuring of cell nitrogen.

(b)  Indirectly, by turbidity.

3.       Determination of cell activity.

(a) Indirectly, by relating the degree of biochemical activity to

the size of the population.

1 a.    Determination of the number of cells directly.

Breed method. A known volume of cell suspension (0.01 ml) is spread uniformly over a glass slide within a specific area (1 sq. cm.). The smear is then fixed, stained, examined under the oil immersion lens, and the cells counted. Since it is impractical to scan the entire area, it is customary to count the cells in a few microscopic fields. The total cell count is determined by calculating the total number of microscopic fields per 1 sq. cm. area of cell suspension. 

Counting chamber method. Special microscope slides are available with chambers designed to contain a cell suspension above an accurately ruled area etched into the glass. The Petroff-Hausser chamber or haemocytometer (because it was originally devised for counting Wood cells) is ruled with squares of known area, and is so constructed that a film of known depth can Be introduced between the slide and the cover slip. Consequently, the volume of the liquid overlying each square is accurately known.

Proportional count method. A standard suspension of particles, for example, plastic beads, where the number of particles per volume is known, is mixed with an equal amount of cell suspension. This mixed suspension is spread on the slide, fixed, and stained. The particles and the cells in each microscopic field are then counted. An average count of the particles and the cell is taken from the number of fields. For example, suppose an average count of 5 parti­cles and 30 cells per field is obtained. If the number of particles in 1 ml of standard suspension is 10,000, then the number of cells per 1 ml of suspension is :

30/5 X 10,000 = 60,000 cells /ml.

In this method there is no need to measure the amount of the suspen­sion spread on the slide.

Electronic counters. An electronic instrument called the Coulter counter can also be used for the direct enumeration of cells in a suspension. Fig. demonstrates the principles of the Coulter counter. The instrument is capable of accurately counting thousands of cells in a few seconds

The suspending fluid, however, must be free of inanimate particles (e. g. dust), since smaller ones will score as cells and larger ones will plug the aperture through which the cells pass.

Direct counting methods are rapid and simple. The morphology of cells can also be observed when they are counted under the microscope. The major disadvantage of these methods is that it gives a total cell count which includes both viable and nonviable cells. Accuracy also declines with very dense and very dilute suspensions because of clumping and statistical errors, respectively. Very dense suspensions, however, can be counted if they are diluted appropriately.

1 b. Determination of the number of cells indirectly by the plate count.

The plate count is based upon the assumption that each organism trapped in or on a nutrient agar medium will multiply and produce a visible colony. The number of colonies therefore are the same as the number of viable cells inoculated into the medium. In this procedure  an appropriately diluted cell suspension is introduced into a petri dish.

Fig.  The plate count technique

An appropriate melted agar medium is poured into the petri dish and is thoroughly mixed with the inoculum by rotating the plate. After the solidification of the medium, the plates are inverted and incubated for 18 to 24 hours. A plate having 30 to 300 colonies is selected for counting the number of organisms.

The plate count has certain disadvantages. If the suspension con­tains different microbial species, then all of them may not grow on the medium used and under the specified conditions of growth. Secondly, if the suspension is not homogeneous and contains aggregates of cells, the resulting colony count will be lower than the actual number of organisms, since each aggregate will produce only one colony. The plate-count technique is used routinely with satisfactory results for the estimation of bacterial population in milk, water, food, and other materials. The method is highly sensitive, i.e., extremely high or very low populations can be counted. However the most obvious advantage of the method is that is counts only living organisms.

Membrane filter count. This method is the same in principle as that of a plate count. A suspension of microorganisms, such as in water or air, is filtered through a millipore filter membrane. The organisms, are retained on the filter disc. The disc is then placed in a petri dish containing a suitable medium. The plates ate incubated and the colonies are observed on the membrane surface. The method has distinct advantages over the plate count. A large volume of the sample can be analyzed, especially when the number of organisms are very few. Secondly various types of microorganisms can be detected by using selective media in the plates and under different conditions of growth.

2 a.   Determination of the cell mass. Measurement of dry weight of cells.

This is the most direct approach for quantitative measurement of a mass of cells. The sample is centrifuged or filtered and the residue or the pellet is washed a number of times to remove all extraneous matter The residue is then dried and weighed. However, it can be used only with very dense cell suspensions. This method is tedious and is applicable mainly in research investigations. It is commonly used for measuring growth of moulds in certain phases of industrial work.

Measurement of cell nitrogen. The major constituent of cell material is protein, and nitrogen is a characteristic constituent of protein. A bacterial population or cell crop can be measured in terms of cell nitrogen. The cells arc to be harvested as described in the first technique, and then the cell nitrogen is estimated by chemical analysis. This is also a tedious method, and can be used only with dense cell suspension.

2 b.   Determination of cell mass.

Turbidity measurements. A most widely used technique of measuring cell mass is by observing the light-scattering capacity of the sample. A suspension of unicellular organisms is placed in a colorimeter or spectrophotometer, and light is passed through it. The amount of the light absorbed or scattered is proportional to the mass of cells in the path of light. When cells are growing exponentially, increase in cell mass is directly related to cell number. This is a rapid and accurate method to estimate dry weight or cell number in unit volume, provided a standard curve is first prepared. A standard curve can be prepared by measuring bacterial growth simulatenously by two methods, and then establishing a relationship between the values obtained. For example, an aliquot of samples is removed from the cell suspension, dried, and the weights per milliliter determined. From the cell suspensions dilutions are prepared, and the organisms are counted by plate-count. At the same time turbidity measurements of the cell suspension are also deter­mined.

Any two sets of the data can then be plotted (cell weights or cell number against turbidity) to obtain a standard curve. For prac­tical purposes, and within certain range of concentrations, a linear or straight-line relationship exists. Thus, by indirectly measuring the turbidity of the suspension, cell weight or cell number can be determined with the help of the standard curve. This method, however, has some limitations. Turbidity is most effective with suspen­sions of moderate density. Suspensions with very high or very low density gives erroneous results. Secondly, it is not possible to measure cultures that are deeply coloured or contain suspended material other than cells. It must be recognized that turbidity measures both living as well as dead cells.

3. Determination of  cell activity.

Measurement of a specific chemical change produced on a constituent of the medium, e.g. acid production from sugar in the nutrient medium. The amount of acid produced is proportional to the magnitude of the cell suspension. Alternatively, specific enzyme may be assayed to measure cell growth.

Metabolism:

            The metabolism of any organism can be divided into two major categories,

1)      Energy generating or degrading patways i.e. Catabolism.

2)      Energy consuming or biosynthetic pathways i.e. Anabolism.

Two biomolecules, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) or nicotenamide adenine dinucleotide phosphate (NADP) are the main link between the two processes of anabolism and catabolism.

            As stated before, metabolism in any organism includes two processes anabolism and catabolism. These two process include all the biochemical reactions of living organism.

1.Anabolism

            It is a process in which essential biomolecules for growth are generated by the utilization of energy. The chief biomolecules required are those of carbon like glucose, ribose, glycerol, pyruvate etc. Some of the biomolecules act as the central metabolic intermediates for all types of carbon and nitrogen compounds required for growth. Some microorganisms can themselves make all the essential organic compounds required for growth as in the case of autotrophs. Such organisms can be grown on simple, chemically defined media. On the other hand, some of the microorganisms which are unable to make most of the organic compounds from atmosphere, are called fastidious organisms. These can only be grown on complex media with different growth factors.

            For the biosynthesis (anabolism) of different essential biomolecules, following anabolic processes take place in organisms:

a)      Synthesis of carbohydrate like glucose, sucrose, cellulose etc.

b)      Synthesis of lipids, glycolipids, phospholipids etc.

c)      Synthesis of amino acids and protein.

d)      Synthesis of nucleic acids.

e)      Synthesis of other growth requirements like vitamins, hormones.

2. Catabolism:

            All the processes in which the nutrients taken in the form of biomolecules as food are broken down (digested) to release energy are called catabolism. Catabolic processes also convert complex organic compounds, stored in the cells of microorganisms (like glycogen granules, polyphosphate, etc) to simpler forms. Each and every organic compound whether it is carbohydrates, protein or fat, can be catabolised according to the requirement of organism. Similar to anabolism certain biomolecules act as link between catabolic and anabolic proceses. A part from these, ATP and NAD(P)H also act as a link between the two types of pathways. The chief catabolic processes involved in cell metabolism are : (a) glycolysis, (b) pentosephosphate pathway (PPP), (c) Ender doudoroff pathway (EDP), (d) tricarboxylic acid cycle (TCA), (e) fermentations, (f) glycoxylate hydrolysis, (g) lipid hydrolysis, (h) protein hydrolysis.

3. Adenosine Triphosphate (ATP)

            ATP is the chief energy carrier molecule in all type of organisms. ATP is required for the anabolic (generative) process of an organism by which organic macromolecules required fro growth are synthesized. ATP is formed by the catabolic (degradative) process in which macromolecules are broken down and energy is generated.

            This energy is the instant energy utilized in the various anabolic processes of the cell. When the ATP is utilized in anbolic process, it is broken down to ADP or AMP releasing phosphate and energy. The ATP utilized in anabolic process is replenished  by catabolic process by reversal reactions. The trapping of chemical energy, released by  the oxidative reactions of the cell, in the form of ATP, is called Phosphorylation. There are three types of Phosphorylation.

(i)Photophosphorylation:  It occurs in the presence of light by photosynthetic organisms mainly in the photoautotrophs and photoheterotrophs.

            In the photosynthetic cells, green pigment (chlorophyll) present  in the chloroplast or bacteriochlorophyll present in the cell membrane of thylakoids, trap energy from solar radiations which become activated, releasing electrons. These electrons then pass through a series of carriers. The energy thus released is trapped in the form of phosphate bonds in ATPs. Photophosphorylation is of two types.

(a)    Cyclic Photophosphorylation:  In this type of Phosphorylation electrons released from chlorophyll molecules (due to excitement by light energy) return back to the same chlorophyll molecules by the same route using electron carriers. Such pathway is cyclic in nature as the electrons pass, energy is released which is traped in the form of ATP.

(b)    Non-cyclic photophosphrylation:  In this type of photophosphorylation electrons released  from chlorophyll molecules do not return back to the same chlorophyll molecules, but instead are received by nicotinamide adenine dinucleotide phosphate (NADP). From NADP electrons enter the electron transport chain wherby oxidative Phosphorylation occurs and ATP is fromed. The electrons released from chlorophyll molecules are replenished in the chlorophyll molecules by photolysis of water.

Thus, we can see that oxygen is released in this type of photophosphorylation. Oxygen is toxic for anaerobes and, therefore, this type of Phosphorylation is not found among anaerobic microorganisms.

(ii)Oxidative Phosphorylation:

            in this Phosphorylation, the electrons collected by certain electron carriers like NAD, NADP and FAD from various sources, are passed into electron transport chain. Finally, electrons reach oxygen or some other inorganic molecule (like iron, nitrate etc), which act as final electron acceptors. The transfer of electrons from one carrier to another released energy, which is used to generate ATP from ADP.

            Oxidative Phosphorylation occurs in the inner memberane of mitochondria in eukaryotes and in plasma membrane of prokaryotes. One molecule of NADPH generates three molecules of ATP, when it enters the electron transport chain for oxidative Phosphorylation, however, one molecule of FADH generates only two molecules of  ATP. The is due to the fact that FADH enters the ETC later than NADPH.

(iii) Substrate level Phosphorylation: In substrate level Phosphorylation, ATP is generated by the transfer of high energy phosphate bond from any other metabolic compound to ADP.

(iv) Nicotinamide Adenine Dinucleotide Phosphate:

            Nicotinamide adenine dinucleotide phosphate (NADP) and Nicotinamide adenine dinucleotide (NAD) are the carriers of electrons (protons) in the cells. Therefore, NADP and NAD serve as the reducing power of the cells in the form of NADP/NAD2 or NADP/NADH2.

                NADP or NAD functions as coenzymes of a large number of oxidoreductase enzyme. They act as electron acceptors during enzymatic removal of hydrogen atoms from specific substrate molecules. Finally, reduced NADP or NAD i.e., NADP H2  or NADH2  release energy. ATP is generated.

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Enzymes

            Enzymes are the biocatalysts found in living organisms which increase the rate of biochemical reactions without being altered themselves. Enzymes are large globular proteins with a molecular weight ranging from 10,000 to millions. Enzymes require optimum conditions of  temperature, pH, substrate concentration for their action. Each enzyme has its own optimum conditions under which they act best.

Mechanism of Enzyme Action

 Like catalysts enzymes also act by lowering down the activation  energy of the reactions.Any reaction occurs when the reactants possess enough energy to attain an activated state called trasition.At this state of high energy bonds break and form anew compound or compounds(product).This state is at the top of energy barrier which has to be overcome so as to complete the reaction.Enzymes combine with the reactions and lower the energy of activation.Thus enzymes enhance the rate of  biochemical reactions by lowering the energy requirement to reach transition state.

     Moreover,enzymes are very specific in their action.This is due to the specific structure of enzymes subtract complex.This mechanism of  enzyme action is called lock and key mechanism of  enzyme action.Thus the absolute structures specificity of enzyme is the cause of their specificity.As each lock has a particular key,in the same way substract has a particular enzyme.The set of events occurring in whole process are as follows(i)Substract with its particular corresponding site attaches to the enzyme and form a enzyme substrate complex,(ii)at the active site high energy state occurs,bonds break and form the products,and(iii)the products get released form the active site and enzyme is recovered as it is.Thus enzyme can react with unlimited number of other reactants.

Enzyme Components

    Generally,enzymes are solely made up of  proteins. But many enzymes also contain a non protein portion, which is necessary for the activity of the enzyme. The protein part of enzyme in these types of enzymes is called apoenzyme and  the nonprotein part is called cofactor.  The cofactor may be a metal ion or an organic molecule (coenzyme). The complete enzyme with both cofactor and apoenzyme is called holoenzyme.

            In the enzyme with metal ion as cofactor are ions such as Mg+2, Fe+2, Fe+3, K+, etc. these may serve as primary catalytic centers, or a bridging  group to bind substrate and enzyme together. Such enzyme with metal ions as cofactor are some times called metalloenzymes e.g. phosphotranferases (which has Mg+2  as metal cofactor), cytochromes and catalase (which have Fe+2 and  Fe+3 metal ion).

The coenzyme , which is a complex organic molecule may be any of the vitamins (trace organic molecule that are vital to the function of all cells and required diet). When the coenzyme is very tightly bound to the enzyme molecules, it is usually called prosthetic group. Some important coenzymes are nicotinamide derivatives, flavin adenine dinucleotide (FAD) derivatives.

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Variations

Spontaneous Mutation

            There is no means to know when and which cell undergo mutation. Any gene of a cell of microorganism is vulnerable to mutation. It is not sure, howeverwhich genewill mutate. Therefore mutation occurs in a gene spontaneously and there is possibility of mutating the genes in a cell and more probability of occurrence of mutant allele in a given population of a microorganism. Spontaneous mutations are  rare ranging from 10-6 to 10-8 per generation depending upon the gene and organism.

            Mutation involves chnges in DNA. Several mechanisms are known that bring about alterations in DNA. These modifications may arise from error in DNA replication, damage to DNA from radiation. Errors occur during replication by substitution of frame shift in DNA sequence.

1.      Substitution Substitution of base pairs is the most common mutation. During replication of DNA repair wrong base pairs are incorporated. Base pair substitutions is of two types, transition and transversion.

Transition Mutation: During replication an incorrect base is correctly hydrogen bonded and incorporated to the template strand. Even the editing system does not recognize it as incorrect. Later on when the base assumes its normal function, the mismatch repair system corrects the mismatching bases at this level. However, if the daughter strand fails to distinguish between the parental and daughter strands. Therefore, the incorrect bases exit in daughter strand and lead to mutation. Such mutation is Known as transition mutation. Transition mutations are common, although most of them are repaired by various proof reading function.

Transversion mutation: Transversion mutation involves the substitution of purine by a pyrimide or a pyrimidine by a purine. This type of mutation is rare due to steric problems of  pairing of purines with purines and pyrimidines with pyrimidines.

2.      FrameShift Mutation

 If there is deletion or insertion of one or few nucleotides in the DNA molecule, this shifts the  reading frame of nucleotide sequences resulting in mutation. Therefore, such mutation that results from shifting in reading frame backward or forward by one or more nucleotide is called frameshift mutation. Generally this mutation occurs where there is a short repeated nucleotide sequence.

INDUCED MUTATION

      Any agent that directly cause damage to the DNA alters the  base sequence or interferes with repair system will certainly induce mutations in DNA of organisms. These agents causing damage to DNA are called mutagens such as chemical.

Chemical mutagens: BASE ANALOGUE: A base analogue is a chemical compound similar to one of the four bases of DNA. It can be incorporated into a growing polynucleotide chain when normal process of replication occurs. These compounds have base pairing properties different  from the bases. They replace the bases and cause stable mutation.

Chemicals changing the Specificity of Hydrogen Bonding

      There are many chemicals that after incorporation into DNA change the specificity of hydrogen-bonding. Those which are used as mutagens are nitrous oxide (HNO2), hydroxylamine (HA) and ethylmethanesulphonate (EMS).

            Nitrous oxide converts the amino group of bases into keto group through oxidative deamination. The order of frequency of deamination (removal of amino group) is adenine>cytosine>guanine.

            Deamination of adenine results in the formation of hypoxanthine, the pairing behaviour of which is like guanine. Hence, it pairs with cytosine instead of thymine replacing AT pairing by GC pairing.

Alkylating Agents

            Addition of an alkyl group to the hydrogen bonding oxygen of guanine (N7 position) and adenine at (N3 position) residues of DNA is done by alkylating agents. As a result of alkylation, possibility of ionization is increased with the introduction of pairing errors. Hydrolysis of linkage of base-sugar occurs resulting in gap in one chain. This phenomenon of loss of alkylated base from the DNA molecule is called depurination

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