Growth of Micro-Organisms: 4 Factors

This article throws light upon the four important physical factors that affect the growth of micro-organisms. The factors are:  1. Temperature 2. Gaseous Requirements 3. Hydrogen Ion Concentration 4. Miscellaneous Physical Requirements.

Factor # 1. Temperature:

Temperature is the most important factor than determines the rates of growth, multiplication, survival, and death of all living organisms. Growth and reproduction of living organisms are dependent on a co-ordinated series of enzyme catalysed chemical reactions.

The rates of enzyme reaction increase with the increase in temperature. Since microbial activity and growth are manifestations of enzymatic reactions, their rates of growth are, temperature-dependent.

In short, temperature determines the rate of growth, the total amount of growth, the metabolic activity, and the morphology of the organisms. Each micro-organism can grow only within a growth temperature range characteristic of the species. The temperature relationships of a micro-organism are usually described by the three cardinal temperatures, the minimum, optimum, and maximum temperatures of growth.

The lowest temperature at which organisms grow is the minimum growth temperature. Most organisms will survive for a varying length of time below this temperature, but will show negligible growth. Minimum growth temperature is difficult to determine precisely, because of an increase in generation time. Growth is not visible until a population of about 1 × 10⁷ cells/ml has been attained.

Whether an organism is capable of growth at a particular temperature depends on the visibility of the growth. The maximum growth temperature is the highest temperature at which growth occurs. A temperature only slightly above this point frequently kills the micro-organisms by inactivating critical enzymes.

Maximum growth temperature is relatively easy to establish, because organisms either grow or are destroyed by high temperature. The optimum temperature is commonly defined as the temperature at which the most rapid rate of multiplication occurs. For most organisms, optimum growth occurs over a temperature range rather than at a fixed temperature.

The optimum temperature is also difficult to agree upon, for the optimum temperature of growth may not be optimum for other cellular activities, for example, maximum acid production or pigment production. Sometimes it also changes the nutritional requirement. Generally the upper limit of the optimum growth temperature is only a few degrees below the maximum growth temperature.

Fig. 18.34 illustrates the effect of temperature on the rate of to bacterial species. Maximum growth temperatures are only 5 to 10 degree higher than the optimum growth temperatures, whereas minimum growth temperatures are approximately 30 degrees lower.

Classification of Bacteria According to Growth Temperature:

The numerical values of the cardinal temperatures (minimum, optimum and maximum), and the range of temperature over which growth is possible, vary widely among bacteria. Some bacteria isolated from not springs are capable of growth at temperature as high as 95°C; others, isolated, from cold environments, can grow at temperature as low as -10°C if a high solute concentration prevents the medium from freezing.

Bacteria are frequently classified into three groups according to their temperature preferences. These groups are not sharply defined, and the distinctions are arbitrary. However, this sort of classification is useful in describing the collective properties of groups of micro-organisms adapted to life in certain environments.

Bacteria are normally classified into three broad groups, psychrophiles, mesophiles, and thermophiles (Table 18.4).

Psychrophiles:

Psychrophilic (Gr. Psychros = cold) bacteria are the predominant organisms in many uncultivated soils, and in lakes, streams, and oceans. They are commonly defined as micro-organisms capable of growth at 0°C, though they grow best at higher temperatures, between 15° to 30°C.

Two groups of psychrophiles have been distinguished:

(1) Obligate psychrophiles cannot grow at temperatures about 19° to 22°C, whereas

(2) Facultative psychrophiles may grow at 30° to 35°C.

Mesophiles:

Most of the commonly studied bacteria are mesophilic (Gr. meso = middle), and these fall into two well defined sub divisions:

(1) Those whose optimum growth temperatures are from 20° to 35° and

(2) Those whose optimum temperatures are between 35° to 45°C.

The first group consists mainly of saprophytes and plant parasites, whereas the second group consists mainly of animal parasites or commensals. Minimum and maximum growth temperatures vary correspondingly, but for the most part and within the range of 10° to 52°C.

Thermophiles:

Thermophiles (Gr. thermo = heat) have optimum growth temperature of 45°C or higher, and generally grow over a range of 40° to 75°C. Two groups of thermophiles have been observed. Obligate thermophiles grow only at high temperatures, usually above 50°C.

Facultative thermophiles grow both at 37°C and 55°C. An organism that is heat resistant, for instance one that withstands pasteurization, but does not grow at high temperatures, is termed thermoduric.

Factor # 2. Gaseous Requirements:

The principal gases that affect microbial growth are oxygen and carbon dioxide. The present atmosphere of the earth contains about 20 per cent (V/V) oxygen. Although almost all higher plants and animals are dependent upon a supply of oxygen, this does not hold true for all micro-organisms. The responses to oxygen among micro-organisms are remarkably variable, and this is an important factor in their cultivation.

The organisms are divided into four groups on the basis of their relationship to molecular oxygen:

(i) Strict or obligate aerobes grow only in the presence of free oxygen.

(ii) Strict or obligate anaerobes grow only in the absence of free oxygen.

(iii)Facultative anaerobes can grow both in the presence and the absence of free oxygen.

(iv) Microaerophilic organisms grow best in the presence of a low concentration of molecular oxygen.

Fig. 18.35 shows the growth pattern of these four groups in deep agar tubes.

Oxygen Requirements:

Molecular oxygen is relatively insoluble in water, and so must be continuously made available to aerobic micro-organisms. Growth of aerobic micro-organisms in tubes or small flasks incubated under normal atmospheric conditions is generally satisfactory. However, when aerobic organisms are to be growth in large quantities, it is advantageous to increase the exposure of the medium to the atmosphere.

This can be accomplished by dispensing the medium in shallow layers, for which suitable containers are available. Alternatively, shaking and bubbling in sterile air or oxygen is done for increasing the availability of oxygen to micro-organisms growing in a liquid medium. However, the amount of oxygen required by various aerobic micro-organisms differs considerably.

Also the amount of oxygen required for maximum growth can differ from that required for other metabolic processes. For example, the amount of oxygen required for the growth of Aspergilus niger is less than that required for the production of citric acid by A. niger.

To cultivate anaerobic micro-organisms special techniques are devised to exclude all atmospheric oxygen from the medium. Anaerobic environment can be established by using one of the following methods.

(i) Addition of reducing compounds, e.g. sodium thioglycollate, cysteine hydrochloride, sodium formaldehyde, sulphoxalate, etc. to the medium to absorb oxygen (Fig. 18.36)

(ii) Mechanical removal of oxygen from an enclosed vessel containing tubes or plates of inoculated medium. The air is pumped out of the vessel and replaced by nitrogen, helium, or a mixture of nitrogen and carbon dioxide (Fig. 18.37)

(iii) Chemical reaction within an enclosed vessel containing the incubated medium, to combine the free oxygen into a compound. This can be as simple as the burning of a small candle or the combustion of small amount of alcohol to use up some of the free oxygen.

A common laboratory method of cultivating an anaerobic micro­organism by introducing pyragallol over the cotton plug in the inoculated slant tube is illustrated in Fig. 18.38.

Carbon Dioxide Requirement:

All micro-organisms utilize carbon dioxide for growth. In some micro-organisms the liberation of carbon dioxide from metabolic reactions is adequate to supply this need. However, when cultures are vigorously aerated, particularly when there is a low cell density, the air may sweep the CO₂ away as quickly as it is produced. Secondly, a sufficient amount of carbon dioxide is to be provided for the cultivation of autotrophs.

In case of autotrophs that can be grown under anaerobic condition, the requirement of CO₂ can be met by providing buffers such as CaCO₃ or NaHCO₃ which release CO₂ when acid is produced by the culture. Carbonates cannot be used in media exposed to air, because the release of CO₂ is rapidly swept away, causing the medium to become extremely alkaline.

Factor # 3. Hydrogen Ion Concentration (pH):

Small size and great mobility of hydrogen ions are of supreme importance in many chemical processes, and more so in biological processes, because of the transfer of hydrogen from one molecule to another. The tendency of hydrogen to dissociate from its original combination thus determines the probability of the reaction.

The concentration of hydrogen is always low in the natural habitat of micro-organisms, but on the other hand the organisms cannot grow in its complete absence. The effect of hydrogen ions is similar to that of metallic ions, high concentration is toxic, moderately low concentration permits growth, and very low concentration is unfavourable for growth.

The acidity of alkalinity of a solution is a function of the relative hydrogen ion (H⁺) concentration or pH which is expressed as the negative log of the hydrogen ion concentration. Microbial growth and activities are strongly affected by the pH of the medium. However, there are wide differences between the pH requirements of the various species.

These differences reflect the normal habits and habitats of the organisms. Micro-organisms show the same type of tolerance to acidity or alkalinity that was observed for temperature. Each species usually shows a range of growth responses to varying pH values, and have a pH optimum for maximal balanced growth.

Organisms which require pH values of 5 or less for maximal growth rate are termed acidophiles, and usually have a pH optimum of 2 or 3. Alkaliphiles grow at pH value between 7 and 12, with the optimum around pH 9.5. Neutrophiles prefer pH values around neutrality (pH 7).

Bacteria, in general, prefer media of pH values near neutrality, and usually cannot tolerate pH values much below 4-5. There are some exceptions to this generalization. The classic example is Thiobacillus thioxidans, which oxidizes sulphur to sulphuric acid, can grow at pH 1.0. Acetic acid bacteria and intestinal bacteria which tolerate the acid of the stomach are other exceptions.

Animal pathogens are usually favoured by an environment at pH 7.2 to 7.4. At the opposite extreme, bacteria that infect the human urinary tract and hydrolyze urea to give ammonia can grow at pH 11. Yeasts prefer slightly acidic media for growth. Moulds prefer more acidic media (pH 4). Many plant and soil micro-organisms, especially Actinomucetes, prefer relatively alkaline conditions.

When micro-organisms are inoculated in a medium originally adjusted to a given pH, it is very likely that this pH will change, depending upon the type of the microbial activity and the composition of the medium. Degradation of proteins and other nitrogenous compounds frequently yields ammonia or other alkaline byproducts; carbohydrate fermentations often produce organic acids.

The change in the pH value brought by such reactions continues until the maximum or minimum pH for the organisms is reached, whereupon the culture dies. The pH of the medium also determines which pathways of metabolism will operate. For example, at an alkaline reaction yeasts ferment glucose to glycerol, whereas at an acid reaction they ferment glucose to ethanol.

Organisms such as Aerobacter aero genes, which can form acetyl methyl-carbinol from glucose, will do so only below pH 6.0. The fate of amino acids in the cell is also decided by pH. At an acid reaction they are decarboxylated to the corresponding amines, whereas at alkaline reaction they are delaminated to an acid.

Buffers are often added to prevent the radical shift in the pH of the medium. Most buffers used in media are mixtures of weakly acidic and weakly alkaline compounds. A combination of K_H2PO₄ and K₂HPO₄ is widely employed in bacteriological media. If micro­organisms from an acid such as acetic acid in a medium buffered with phosphate, a part of the basic salt (K₂HPO₄) is converted to the weakly acidic salt.

K₂HPO₄ + HC₂H₃O₂←→ KH₂PO₄ + KC₂H₃O₂

The pH of the medium falls only slightly. Conversely, a basic microbial product reacts with the acidic salt (KH₂PO₄) to form a dibasic compound that is only weakly alkaline. Many culture media contain amphoteric substances such as peptones. These compounds possess both amino and carboxyl radicals, which can dissociate as basic and acidic groups.

Insoluble carbonates such as CaCO₃ and MgCO₃ are also added to media to prevent a drop in pH as acid is produced. Being insoluble, they have no direct effect on pH, but when acid is formed and the reaction falls below pH 7.0, the carbonate decomposes, CO₂ is evolved, and the acid is converted to its calcium or magnesium salt.

The extent to which a medium should or may be buffered depends on its intended purpose, and is limited by the buffering capacity of the compounds used. Some large fermentation apparatuses are equipped with automatic controls that maintain a constant pH.

Factor # 4. Miscellaneous Physical Requirements:

Additional physical factors are to be considered for the growth of certain fastidious organisms. For example photosynthetic micro-organisms (alagae, photosynthetic bacteria) must be exposed to a source of illumination, since light is their source of energy.

Halophiles and osmophiles isolated from sea and other natural bodies of water of high salinity can grow only when the medium contains an unusually high concentration of salt.

The successful cultivation of micro-organisms in the laboratory is based upon two basic principles; nutritional requirement to prepare a suitable nutrient medium, and appropriate physical conditions to obtain maximum growth.