Factors affecting microbial spoilage of pharmaceutical products

Factors affecting microbial spoilage of pharmaceutical products

By understanding the influence of environmental parameters on microorganisms, it may be possible to manipulate formulations to create conditions which are as unfavorable as possible for growth and spoilage, within the limitations of patient acceptability and therapeutic efficacy.

Types and size of contaminant inoculum

When failures inevitably occur from time to time, knowledge of the microbial ecology and careful identification of contaminants can be most useful in tracking down the defective steps in the design or production process. Low levels of contaminants may not cause appreciable spoilage, if unable to replicate in a product; however, an unexpected surge in the contaminant bioburden may present an unacceptable challenge to the designed formulation. This could arise as if there was a lapse in the plant-cleaning protocol; a biofilm detached itself from within supplying pipework; or the product had been grossly misused during administration. Inoculum size alone is not always a reliable indicator of likely spoilage potential. Low levels of aggressive pseudomonads in a weakly preserved solution may suggest a greater risk than tablets containing fairly high numbers of

fungal and bacterial spores.

When an aggressive microorganism contaminates a medicine, there may be an appreciable lag period before significant spoilage begins, the duration of which decreases disproportionately with increasing contaminant loading. As there is usually a considerable delay between manufacture and administration of factory-made medicines, growth and attack could ensue during this period unless additional steps were taken to prevent it.

The isolation of a particular microorganism from a markedly spoiled product does not necessarily

mean that it was the initiator of the attack. It could be a secondary opportunist contaminant which had overgrown the primary spoilage organism once the physicochemical properties had been favourably modified by the primary spoiler.

Nutritional factors

Microorganisms enable them to utilize many formulation components as substrates for biosynthesis and growth. The use of crude vegetable or animal products in a formulation provides an additionally nutritious environment. Even demineralized water prepared by good ion-exchange methods will normally contain sufficient nutrients to allow significant growth of many waterborne Gram-negative bacteria such as Pseudomonas spp. When such contaminants fail to survive, it is unlikely to be the result of nutrient limitation in the product but due to other, non-supportive, physicochemical or toxic properties.

Acute pathogens require specific growth factors normally associated with the tissues they infect but which are often absent in pharmaceutical formulations. They are thus unlikely to multiply in them, although they may remain viable and infective for an appreciable time in some dry products where the conditions are suitably protective.

Moisture content: water activity (A_w)

Microorganisms require readily accessible water in appreciable quantities for growth to occur. By measuring a product’s water activity (A_w), it is possible to obtain an estimate of the proportion of uncomplexed water that is available in the formulation to support microbial growth, using the formula: A_w = vapour pressure of formulation/vapour pressure of water under similar conditions.

The greater the solute concentration, the lower is the water activity. With the exception of halophilic bacteria, most microorganisms grow best in dilute solutions (high A_w) and, as solute concentration rises (lowering A_w), growth rates decline until a minimal growth-inhibitory Aw, is reached.

The Aw of aqueous formulations can be lowered to increase resistance to microbial attack by the addition of high concentrations of sugars or polyethylene glycols. Aw can also be reduced by drying, although the dry, often hygroscopic medicines (tablets, capsules, powders) will require suitable packaging to prevent resorption of water and consequent microbial growth.

Condensation similarly formed on the surface of viscous products such as syrups and creams, or exuded by syneresis from hydrogels, may well permit surface yeast and fungal spoilage.

Redox potential

The ability of microbes to grow in an environment is influenced by its oxidation-reduction balance (redox potential), as they will require compatible terminal electron acceptors to permit their respiratory pathways to function. The redox potential even in fairly viscous emulsions may be quite high due to the appreciable solubility of oxygen in most fats and oils.

Storage temperature

Spoilage of pharmaceuticals could occur potentially over the range of about -20°C to 60°C, although it is much less likely at the extremes. The particular storage temperature may selectively determine the types of microorganisms involved in spoilage. A deep freeze at -20°C or lower is used for long-term storage of some pharmaceutical raw materials and short-term storage of dispensed total parenteral nutrition (TPN) feeds prepared in hospitals. Reconstituted syrups and multi-dose eye-drop packs are sometimes dispensed with the instruction to ‘store in a cool place’ such as a domestic fridge (8°–12°C), partly to reduce the risk of growth of contaminants inadvertently introduced during use. Conversely, Water for Injections (EP) should be held at 80°C

or above after distillation and before packing and sterilization to prevent possible regrowth of Gram negative bacteria and the release of endotoxins.

pH

Extremes of pH prevent microbial attack. Around  neutrality, bacterial spoilage is more likely, with reports of pseudomonads and related Gram-negative bacteria growing in antacid mixtures, flavoured mouthwashes and in distilled or demineralized water. Above pH 8 (e.g. with soap-based emulsions) spoilage is rare. In products with low pH levels (e.g. fruit juice-flavoured syrups with a pH 3–4), mould or yeast attack is more likely. Yeasts can metabolize organic acids and raise the pH to levels where secondary bacterial growth can occur. Although the use of low pH adjustment to preserve foodstuffs is well established (e.g. pickling, coleslaw, yoghurt), it is not practicable to make deliberate use of this for medicines.

Packaging design

Packaging can have a major influence on microbial stability of some formulations in controlling the entry of contaminants during both storage and use. Considerable thought has gone into the design of containers to prevent the ingress of contaminants into medicines for parenteral administration, owing to the high risks of infection by this route. Self-sealing rubber wads must be used to prevent microbial entry into multi-dose injection containers following withdrawals with a hypodermic needle.

Where medicines rely on their low A_w to prevent spoilage, packaging such as strip foils must be of water vapour-proof materials with fully efficient seals. Cardboard outer packaging and labels themselves can become substrates for microbial attack under humid conditions, and preservatives are often included to reduce the risk of damage.

Protection of microorganisms within pharmaceutical products

The survival of microorganisms in particular environments is sometimes influenced by the presence of relatively inert materials. Thus, microbes can be more resistant to heat or desiccation in the presence of polymers such as starch, acacia or gelatin. Adsorption onto naturally occurring particulate material may aid establishment and survival in some environments. There is a belief, but limited hard evidence, that the presence of suspended particles such as kaolin, magnesium trisilicate or aluminium hydroxide gel may influence contaminant longevity in those products containing them, and that the presence of some surfactants, suspending agents and proteins can increase the resistance of microorganisms to preservatives, over and above their direct inactivating effect on the preservative itself.

Classification and Nomenclature of Enzymes by the Reactions they Catalyze

Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)

Enzyme Nomenclature

1. General principles

Because of their close interdependence, it is convenient to deal with the classification and nomenclature together.

The first general principle of these 'Recommendations' is that names purporting to be names of enzymes, especially those ending in -ase, should be used only for single enzymes, i.e. single catalytic entities. They should not be applied to systems containing more than one enzyme. When it is desired to name such a system on the basis of the overall reaction catalyzed by it, the word system should be included in the name. For example, the system catalyzing the oxidation of succinate by molecular oxygen, consisting of succinate dehydrogenase, cytochrome oxidase, and several intermediate carriers, should not be named succinate oxidase, but it may be called the succinate oxidase system. Other examples of systems consisting of several structurally and functionally linked enzymes (and cofactors) are the pyruvate dehydrogenase system, the similar 2-oxoglutarate dehydrogenase system, and the fatty acid synthase system.

In this context it is appropriate to express disapproval of a loose and misleading practice that is found in the biological literature. It consists in designation of a natural substance (or even of an hypothetical active principle), responsible for a physiological or biophysical phenomenon that cannot be described in terms of a definite chemical reaction, by the name of the phenomenon in conjugation with the suffix -ase, which implies an individual enzyme. Some examples of such phenomenase nomenclature, which should be discouraged even if there are reasons to suppose that the particular agent may have enzymic properties, are: permease, translocase, reparase, joinase, replicase, codase, etc..

The second general principle is that enzymes are principally classified and named according to the reaction they catalyze. The chemical reaction catalyzed is the specific property that distinguishes one enzyme from another, and it is logical to use it as the basis for the classification and naming of enzymes.

Several alternative bases for classification and naming had been considered, e.g. chemical nature of the enzymes (whether it is a flavoprotein, a hemoprotein, a pyridoxal-phosphate protein, a copper protein, and so on), or chemical nature of the substrate (nucleotides, carbohydrates, proteins, etc.). The first cannot serve as a general basis, for only a minority of enzymes has such identifiable prosthetic groups. The chemical nature of the enzyme has, however, been used exceptionally in certain cases where classification based on specificity is difficult, for example, with the peptidases (subclass EC 3.4). The second basis for classification is hardly practicable, owing to the great variety of substances acted upon and because it is not sufficiently informative unless the type of reaction is also given. It is the overall reaction, as expressed by the formal equation that should be taken as the basis. Thus, the intimate mechanism of the reaction, and the formation of intermediate complexes of the reactants with the enzyme is not taken into account, but only the observed chemical change produced by the complete enzyme reaction. For example, in those cases in which the enzyme contains a prosthetic group that serves to catalyze transfer from a donor to an acceptor (e.g. flavin, biotin, or pyridoxal-phosphate enzymes) the name of the prosthetic group is not normally included in the name of the enzyme. Nevertheless, where alternative names are possible, the mechanism may be taken into account in choosing between them.

A consequence of the adoption of the chemical reaction as the basis for naming enzymes is that a systematic name cannot be given to an enzyme until it is known what chemical reaction it catalyzes. This applies, for example, to a few enzymes that have so far not been shown to catalyze any chemical reaction, but only isotopic exchanges; the isotopic exchange gives some idea of one step in the overall chemical reaction, but the reaction as a whole remains unknown.

A second consequence of this concept is that a certain name designates not a single enzyme protein but a group of proteins with the same catalytic property. Enzymes from different sources (various bacterial, plant or animal species) are classified as one entry. The same applies to isoenzymes (see below). However, there are exceptions to this general rule. Some are justified because the mechanism of the reaction or the substrate specificity is so different as to warrant different entries in the enzyme list. This applies, for example, to the two cholinesterases, EC 3.1.1.7 and 3.1.1.8, the two citrate hydro-lyases, EC 4.2.1.3 and 4.2.1.4, and the two amine oxidases, EC 1.4.3.4 and 1.4.3.6. Others are mainly historical, e.g. acid and alkaline phosphatases (EC 3.1.3.1 and EC 3.1.3.2).

A third general principle adopted is that the enzymes are divided into groups on the basis of the type of reaction catalyzed, and this, together with the name(s) of the substrate(s) provides a basis for naming individual enzymes. It is also the basis for classification and code numbers.

Special problems attend the classification and naming of enzymes catalyzing complicated transformations that can be resolved into several sequential or coupled intermediary reactions of different types, all catalyzed by a single enzyme (not an enzyme system). Some of the steps may be spontaneous non-catalytic reactions, while one or more intermediate steps depend on catalysis by the enzyme. Wherever the nature and sequence of intermediary reactions is known or can be presumed with confidence, classification and naming of the enzyme should be based on the first enzyme-catalyzed step that is essential to the subsequent transformations, which can be indicated by a supplementary term in parentheses, e.g. acetyl-CoA:glyoxylate C-acetyltransferase (thioester-hydrolysing, carboxymethyl-forming) (EC 2.3.3.9, cf. section 3).

To classify an enzyme according to the type of reaction catalyzed, it is occasionally necessary to choose between alternative ways of regarding a given reaction. Some considerations of this type are outlined in section 3 of this chapter. In general, that alternative should be selected which fits in best with the general system of classification and reduces the number of exceptions.

One important extension of this principle is the question of the direction in which the reaction is written for the purposes of classification. To simplify the classification, the direction chosen should be the same for all enzymes in a given class, even if this direction has not been demonstrated for all. Thus the systematic names, on which the classification and code numbers are based, may be derived from a written reaction, even though only the reverse of this has been actually demonstrated experimentally. In the list in this volume, the reaction is written to illustrate the classification, i.e. in the direction described by the systematic name. However, the common name may be based on either direction of reaction, and is often based on the presumed physiological direction.

Many examples of this usage are found in section 1 of the list. The reaction for EC 1.1.1.9 is written as an oxidation of xylitol by NAD⁺, in parallel with all other oxidoreductases in subgroup EC 1.1.1, and the systematic name is accordingly, xylitol:NAD⁺ 2-oxidoreductase (D-xylulose-forming). However, the common name, based on the reverse direction of reaction, is D-xylulose reductase.

2. Common and Systematic Names

The first Enzyme Commission gave much thought to the question of a systematic and logical nomenclature for enzymes, and finally recommended that there should be two nomenclatures for enzymes, one systematic, and one working or trivial. The systematic name of an enzyme, formed in accordance with definite rules, showed the action of an enzyme as exactly as possible, thus identifying the enzyme precisely. The trivial name was sufficiently short for general use, but not necessarily very systematic; in a great many cases it was a name already in current use. The introduction of (often cumbersome) systematic names was strongly criticized. In many cases the reaction catalyzed is not much longer than the systematic name and can serve just as well for identification, especially in conjunction with the code number.

The Commission for Revision of Enzyme Nomenclature discussed this problem at length, and a change in emphasis was made. It was decided to give the trivial names more prominence in the Enzyme List; they now follow immediately after the code number, and are described as Common Name. Also, in the index the common names are indicated by an asterisk. Nevertheless, it was decided to retain the systematic names as the basis for classification for the following reasons:

(i) The code number alone is only useful for identification of an enzyme when a copy of the Enzyme List is at hand, whereas the systematic name is self-explanatory;

(ii) The systematic name stresses the type of reaction; the reaction equation does not;

(iii) Systematic names can be formed for new enzymes by the discoverer, by application of the rules, but code numbers should not be assigned by individuals;

(iv) Common names for new enzymes are frequently formed as a condensed version of the systematic name; therefore, the systematic names are helpful in finding common names that are in accordance with the general pattern.

It is recommended that for enzymes that are not the main subject of a paper or abstract, the common names should be used, but they should be identified at their first mention by their code numbers and source. Where an enzyme is the main subject of a paper or abstract, its code number, systematic name, or, alternatively, the reaction equation and source should be given at its first mention; thereafter the common name should be used. In the light of the fact that enzyme names and code numbers refer to reactions catalyzed rather than to discrete proteins, it is of special importance to give also the source of the enzyme for full identification; in cases where multiple forms are known to exist, knowledge of this should be included where available.

When a paper deals with an enzyme that is not yet in the Enzyme List, the author may introduce a new name and, if desired, a new systematic name, both formed according to the recommended rules. A number should be assigned only by the Nomenclature Committee of IUBMB.

The Enzyme List contains one or more references for each enzyme. It should be stressed that no attempt has been made to provide a complete bibliography, or to refer to the first description of an enzyme. The references are intended to provide sufficient evidence for the existence of an enzyme catalyzing the reaction as set out. Where there is a major paper describing the purification and specificity of an enzyme, or a major review article, this has been quoted to the exclusion of earlier and later papers. In some cases separate references are given for animal, plant and bacterial enzymes.

3. Scheme for the classification of enzymes and the generation of EC numbers

The first Enzyme Commission, in its report in 1961, devised a system for classification of enzymes that also serves as a basis for assigning code numbers to them. These code numbers, prefixed by EC, which are now widely in use, contain four elements separated by points, with the following meaning:

(i) The first number shows to which of the six main divisions (classes) the enzyme belongs,

(ii) The second figure indicates the subclass,

(iii) The third figure gives the sub-subclass,

(iv) The fourth figure is the serial number of the enzyme in its sub-subclass.

The subclasses and sub-subclasses are formed according to principles indicated below.

The main divisions and subclasses are:

No.	Class	Type of reaction catalyzed
1	Oxidoreductases	Transfer of electrons (hydride ions or H atoms)
2	Transferases	Group transfer reactions
3	Hydrolases	Hydrolysis reactions (transfer of functional groups to water)
4	Lyases	Addition of groups to double bonds, or formation of double bonds by removal of groups
5	Isomerases	Transfer of groups within molecules to yield isomeric forms
6	Ligases	Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to ATP cleavage

Mass Spectroscopy

MASS SPECTROSCOPY

- Branch of Spectroscopy

- Analytical technique that gives information concerning the molecular structure the of organic and inorganic compounds

- It can determine molecular weight of as high as 4000

- Qualitative analytical tool to characterize different organic substances

- The quantitative analysis of mixtures (gases or liquids and sometimes solids)

- Based on the simple principle

- Yet it is a very complex and very expensive instrument

- Commonly used because of its high speed and reliability

PRINCIPLE

The compounds under investigation are bombarded with a beam of electrons which produce an ionic molecules or ionic fragments of the original species then they are separated on the basis of the difference in their masses. Suppose ionization is as follows;

M + e^- ® M⁺ + 2e^-

Where:

M⁺ = an ionized molecule

e^- = an electron

The ions are then accelerated in an electric field at voltage V. Now the energy given to each particle is eV and this is equal to the kinetic energy of the ions.

½ mn² = e V

Or n = Ö 2e V/m

Where:

n = velocity of the particle of mass, m

e = charge on an electron

m = mass of the particle

V = accelerating voltage

All the particles possess the same energy, eV. Also, all particles have the same kinetic energy, ½ mn². As the value of m varies from particle to particle, the velocity of particle in motion also changes to meet the potential energy. E.g. For a particle with mass, m₁ and n₁

½ m₁n₁²= eV

Similarly, for particles of mass m₂, m₃, m₄,………………..m_n the velocities are n₁, n₂, n₃, …………….n_n. Now their kinetic energies are:

½ m₂n₂²= eV ½ m₃n₃²= eV ½ m₄n₄²= eV………………½ m_nn_n²= eV

From above, we have;

½ m₂n₂²= ½ m₃n₃²= ½ m₄n₄²= ………………½ m_nn_n²= eV

After the charged particles have been accelerated in an applied voltage, they enter into the magnetic field, H. This field attracts the particles and move in a circular around it. The attractive force due to magnetic field is Hen, where the balancing centrifugal force on the particle is mn²/r. When the particle starts moving uniformly around the circular path the two forces become equal. i.e.

mn²/r = H.e.n

r = mn / H.e

Where, r is the radius of circular path of the particle in the motion.

From above equations;

r = [m / H.e] Ö 2eV/m

On squaring both sides;

r² = [m² / H².e²] 2eV/m

\ m/e = [H² . r²]/ 2V

The radius of circular path of the particles depends on the accelerating voltage- V, the magnetic field-H and the ratio of mass upon charge-m/e. As e, V and H are constants; the radius of ionized molecule depends on the mass only which is actually the main basis of separation of particles.

INSTRUMENTATION

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.