APPLICATIONS IN THE FOOD INDUSTRY AND AGRICULTURE.
OTHER FIELDS OF UTILIZATION: Here, reference will be made to the possibility for the quick and efficient destruction in an ozone atmosphere of various non-pathogenic micro-organisms including molds, spores and other primitive single cell creatures. The wide ranging possibilities for using ozone in the food industry and agriculture as well as in other fields, are created similarly by it's bacterial and germ killing power. Not only does it act as a germicide but as a spore killing agent as well. Fruits, foodstuffs, etc., exposed to its effect, under go a more or less pronounced change as a consequence of it's action on the vital process of cells, the process of there metabolism particularly, through the inactivation of their metabolic products. At the same time it reacts with other materials present that can be oxidized and thereby it destroys fragrances and odors. Utilization of these properties makes ozone eminently suitable for increasing the storage life of perishable foods in refrigerated premises. At the same time it's use is economic as the investment and operational costs of the equipment are on a acceptable level in relation to the size of refrigerated rooms. It's application eliminates the risk of leaving the unpleasant odor or other traces of antiseptics used for preservation on food stuffs. Utilization of ozone for increasing the storage life of food, particularly if held at low temperatures, is believed to have started in 1909 when, in the cold storage plant of cologne, the reduction in the germ count on the surface of meat stored there was observed after an ozone generator had been installed in the duct of fresh air used to ventilate the storage room. Much more extensive examinations and experiments were required on the storage of fruits in cold storage plants in order to decide whether treatment by ozone could be deemed favourable or unfavourable because of the different requirements imposed on the storage of various fruits. Although few publications or research reports have as yet become part of the public domain, the use of ozone is increasing in several major cold storage plants in Europe. Van Laer and Troquet described, as early as 1928, the utilization of ozone in breweries. R.I. Tenny focused attention again 1972 on the possibilities for it's use in the brewing industry. The technical shortcomings of the ozone generators in the 1940's were responsible for the setbacks encountered at the time.
PRESERVATION AND STORAGE Practical operations for preservation start with the sterilization of air in such a way that air entering the storage room contains a sufficient amount of ozone to destroy micro-organisms. At the same time, however, ozone decomposition to a significant extent is to be expected due to the high moisture content required, The walls of the storage room, the packaging materials, the absorption effect of the stored goods, and also to the oxidation reactions taking place. These two requirements demand the most perfect distribution of ozonated air in the storage room and make it imperative that the capacity of the ozone generator ensures the maintenance of the appropriate ozone concentration throughout the whole mass of air. Otherwise it may happen that ozone will not reach the storage space properly, let alone the surface of the goods stored. The required effect can be attained by a strong air movement, the storage space, in turn , need not be hermetically sealed as, for example, in the case of storage under static CO2 gas atmosphere. A state of equilibrium can set in, even in these relatively closed premises, between the amount of ozone consumed by the environment, the packaging materials and the walls etc., through absorption, and utilized by the stored goods (for the destruction of surface germs, the oxidation of metabolic products etc.) on the one hand, and the amount of ozone introduced on the other. After stopping feed, decomposition continues for which ozone is supplied up to a certain time by desorption from the environment; even so, the total depletion of the ozone content sets in rapidly. During storage, ozone exerts a threefold effect by destroying the micro-organisms, oxidizing the odors and effecting the processes of metabolism.
GERMICIDAL EFFECT This effect of ozone has already been covered in general terms. For applications in the food industry, a greater emphasis should understandably be put on the changes in quality taking place following the ozone treatment, along with the specific effects exerted on individual products. The germicidal power of ozone is generally specific in respect of individual species. It's primary action on molds is to suppress there growth and this effect can set in rapidly, particularly in the initial stage on a mold free surface. Afterwards this process leads to the destruction of cultures already formed. Ozone attacks immediately the easily accessible cells on the surface since ozone exerts a surface effect in the first place and has a only slight depth of penetration. Kolodyaznaya and Sponina investigated the micro flora causing the deterioration of the potato. Pure mold cultures of Fusarium Solani, Rhisoctonia Solani, and Phytophtora Solani were exposed to the action of ozone. From these species Fusarium Solani proved to be resistant to ozone. Ozonization applied for the storage of refrigerated meat destroys surface micro-organisms, particularly the family of Pseudomonas responsible for spoilage. Increasing the moisture content of the environment favourably influences the germicidal effect. This is brought about by the swelling of microbes making them more susceptible to destruction. Experiments conducted with beef showed that ozone is most efficient if the surface has a definite moisture content of around 60%.
EFFECT ON ODORS Ozone itself has a characteristic odor, yet the result of application does not mask odors. Atomic oxygen formed by decomposition of ozone immediately oxidizes the differently smelling materials. The characteristic putrid odor, however, remains and is difficult to eliminate even with the use of ozone. In general, the lower the temperature and the larger the molecules taking part in the reaction, the weaker is the oxidizing effect. The moisture content in the air has no effect on the process. At very slight concentration, say between 0.01 and 0.04 ppm. ozone, the air of the room of storage space is felt to be fresh and pleasant and no stuffy odor is sensed any more. It is an established fact that the odor of aromatic fruits such as strawberries is enhanced in the presence of ozone. It is possible that the formation of fragrances and odors giving the fruit its characteristic flavour is assisted by ozone. The sterilization of the air in fruit stores by ozone prevents the odors of packaging materials from being transferred to the goods stored, a phenomena which frequently takes place otherwise, particularly when wooden crates are used in refrigerated stores at relative humidity of 85% to 90%
EFFECT ON METABOLISM The effect on metabolism is also a consequence of the strong oxidizing power of ozone. no deterioration of fruit was observed, but the reason for this is that ozone only affects the surface of fruit which contain compounds difficult to oxidize in most cases. During storage the process of respiration of fruit is speeded up and so is ripening. In the case of a more rapid ripening than would be desirable, ethylene is produced which affects the other fruit and so initiates even more intensive ripening. The external signs of this process are the turning brown of the skin, the softening of the flesh of the rest of the fruit and, finally, decay. This process is controlled by the presence of ozone because it oxidizes the metabolic products created initially, reducing thereby the process of back action on other fruits. Moreover, it promotes the healing of wounds and enhances resistance to further infection.
MEAT For the storage of meat it was found that a satisfactory effect can be brought about by one or two periods daily of ozone application, lasting two hours each time if the ozone content was held at 6 mg. (0)m. (air). Application of ozone proved to be particularly beneficial to the process of tenderizing meats. During tenderizing, fresh beef sides are kept for 42 to 44 hours in a closed space at a temperature of 293K and a relative humidity of 85%. The process of tenderization consists, in actual fact, of the digestive action caused by enzymes naturally present to soften and slacken muscles and connective tissue. The same process can take as much as 20 days at a temperature of 279K. The accelerating effect of temperature increases on tenderization promotes the formation of fertile soil suitable for the multiplication of infectious bacteria and spores of deleterious nature. The aim to be achieved with ozone treatment is the destruction of these harmful surface organisms. In such a tenderizing room a concentration of 0.1 ppm. and a relative humidity of 60% to 90% should be maintained, according to Ewell. According to others, ozone is efficient, even if present in a concentration of 0.04 ppm. and, although it fails to bring about full sterilization, it still retards the growth of bacteria. The germicidal action of ozone is restricted only to the surface in the case of meat too, and has a small depth of penetration. Molds present in the form of spores can be destroyed only if attacked by a high concentration of ozone. The storage life of beef in a refrigerated state can be increased by 30% to 40% if the beef is kept in a atmosphere of 10 to 20 mg. (0) m (air) and the microbial saturation of its surface is not greater than 10 bacteria cm. Billion conducted a detailed investigation on the storage life of beef, veal, lamb, pork, chicken, and rabbit in ozonized atmospheres. In the case of the varieties of meat stored in a normal atmosphere, it was found that a significant microbial contamination sets in after 7 days. Contamination's of the same level were reached on meats exposed to the action of ozone only after 14 days under identical conditions. It can be stated in general that, in a refrigerated atmosphere and in the presence of ozone, the growth of the surface micro flora (Pseudomonas families, spores, salmonellae, staphylococci) is slowed down. Nevertheless, no effect is exerted by ozone on the surface micro flora if the extent of contamination is large already. Thus, although ozone fails to produce an express antiseptic effect on stored meats, it still makes the atmosphere of refrigerated stores fresh and healthy. Freshly caught fish can be stored longer if washed in water containing ozone; If it is preserved by ice produced from ozone containing water a higher increase in damage-free storage time can be obtained.
CHEESE Experiments for the use of ozone during the process of ripening and storage of cheese were successfully conducted. Spores created on the surfaces of cheese during the ripening period were destroyed and storage life was increased to 11 weeks by the application of a small ozone concentration (0.02 ppm.) at 288K and a relative humidity of 80% to 85%. Experiments were conducted on cheddar cheese indicating that odors otherwise present in storage rooms were also eliminated by the oxidizing action of ozone.
EGGS Ozone has been successfully used for the storage of eggs. By the end of the 1930s, more than 80% of refrigerated egg stores in the United States were equipped with ozone generating equipment to increase storage life.
BEVERAGES Ozone treatment speeds up the aging of wine, avoids turbidity, and refines its bouquet, which is retained for a longer time. The storage life of milk, bottled juices and soft drinks is also improved by ozone through suppressing sour spoilage. Independently of municipal water supply, large quantities of bottled water disinfected with ozone are retailed in the United States. The sterilization of water required for the production of beverages is a significant subject due to the increased demand for water of good quality by production plants. For the industrial scale production of soft drinks, so-called humic waters can be used which are perfect in meeting biological requirements (taste, odor), but their brownish color is undesirable. This color taken from the soil can be eliminated by means of ozone. The main requirement to be met is retaining of the sterile state in the 4 to 8 week period elapsing between filling and consumption. Ozone has proved to be the most efficient method for sterilization surpassing conventional processes used previously such as UV, irradiation, chlorination, silver treatment, sterilization filtering. One of the most widely consumed soft drinks is bottled mineral water. Many grades, however, contain manganese and iron. If the usual methods are applied for the removal of manganese and iron, the naturally dissolved carbon dioxide content is largely depleted. Use of ozone in this respect is of particular advantage as both iron and manganese can be fully oxidized by ozone with the simultaneous retention of the high concentration of the dissolved carbon dioxide. Ozone is also used in the milk industry, to suppress souring. Such a sterilization step greatly increases storage life. In the brewing industry, ozone can be used with advantage for the disinfection of pipe lines, filters, bottles, etc. If phenolic wastes are present, chlorine oxidizes them to chlorophenols, which have tastes and odors intolerable to brewers. ozone creates special points of special interest to brewers and users of other similar processes because it represents a biocide with non-persistent and non-toxic residues.
DISINFECTION AND REMOVAL OF ODORS Storage places, warehouses and refrigerated stores can be disinfected in most cases by the admission of ozonized air. This is independent of the direct action exerted on food, fruits, beverages, etc. stored there. Such a process, apart from disinfection, removes the frequently unpleasant odors of packaging materials so the various produces retain there original flavour. It happens frequently that the stored products, due to insufficient air locking, imperfect separation or facilities for communication. The oxidation of compounds creating odors in such premises has the advantage that it creates an atmosphere resembling pleasant fresh air. For such a purpose a very low ozone concentration of 0.01 to 0.04 ppm. is sufficient. In refrigerated tunnels, meat tenderizing halls and meat warehouses, ozone is generated by special sterilization lamps. These are designed to have a portion of there radiation band in the range below 200nm. so they have an active photochemical ozone generation effect. Initially, when by product ozone was discovered in the application of UV lamp systems, efforts were made to restrict the formation of traces of ozone associated with the use of these lamps. Later it was found that the presence of ozone at concentrations of up to 0.1 ppm. by volume protects meat from decay. The increase in storage life (see preceding section) had the secondary benefit of reducing odors. The effect of ozone in a domestic refrigerators was also investigated. Here to, sterilizing lamps were used for ozone generation. It was necessary to find a balance between the amount of ozone potentially available and ozone demand that mold and bacteria on the food and the refrigerator walls would be destroyed and the level of food odor and there transfer to other foods would be greatly reduced. Ozone, if present at a concentration of 0.1 ppm. is capable of destroying micro-organisms and removing odors after an exposure lasting about 48 hours. A longer exposure time (At lower concentrations) is equivalent in terms of bactericidal effect; it fails, however to eliminate the odors.
APPLICATION FOR AGRICULTURE Ozone is eminently suitable for the processing of various by-products and secondary products originating in the agriculture and food industries. It has proved suitable for the bleaching of bees wax, starch, flour, straw products, bones, feathers, lard among others. The majority of these products become whiter after treatment and there smell is improved. If cotton and wool are treated with ozone, the grease and wax like materials on the surfaces of fibres are decomposed. The removal of these substances increases significantly the storage life of cotton fibre and improves the dye ability of wool with a simultaneous bleaching action. The Ozonization of flax speeds up the process of ripening and facilitates further processing. For the removal of odors from large stalls, ozone has long been used in the Soviet Union, the United States and Canada, in conjunction with the process described previously.
OTHER FIELDS OF APPLICATION Finally, fields of application that have not been referred to previously will be summarized. At the same time, the processes and methods described previously will serve as examples for a variety of further applications where each of them uses a high reactivity of ozone in some form. The textile industry uses ozone to bleach various basic materials, yarns and textiles. Health protection or, in an indirect way, medical science examined long ago the disinfecting and bacterial action of ozone in the interest of its use on a wide basis. In this framework, successful experiments were conducted for the bleaching of hospital linen with ozone generated via sterilizing lamps. Orlowski reports on the disinfection of bandages and surgical devices where the action of ozone was used instead of the conventional temperature effect. Chemical industry and metallurgy utilize the process described previously for the neutralization of vent gases from sewage works, to remove industrial odors which are frequently harmful to health. For deodorization, of refresh of air in rooms accommodating large masses of people such as theatres, assembly halls, etc., and for improving air in offices, ozonized air is used widely in conjunction with air conditioning systems. In this way, demand for make-up air is reduced as the recycle system furnishes air of sufficient purity. For premises such as theatres, assembly halls, etc., stringent code requirements limit the allowable concentration of ozone in the air; this concentration must not exceed several ppm. generally. At the same time it has been proved that the effect of various unpleasant smells and body odor, cigarette smoke, etc., can be eliminated in practice by treatment with a small amount of ozone.
SWIMMING POOLS In the field of the classical water and wastewater treatment, further new applications have been found for ozone processes. According to official regulations, only hygienically and aesthetically perfect water is allowed to be used in baths and swimming pools due to infection hazard. Ozone as a dominating disinfectant, is not only efficient in reconditioning bathing water due to the destruction of spores and viruses, as well as the decomposition of human urine, but its use brings economies owing to a reduction in the demand for make-up water. Equipment normally used to purify drinking water complemented by special units required for the application is suitable also for the reconditioning of bathing water. (Reconditioning means an appropriate recirculation method to ensure proper filtering and dispersion of the disinfectant). In Switzerland, hundreds of indoor and outdoor swimming pools are equipped with these ozone treatment devices today. The development of the "Sauter-Var Ozone Process" is due to the Swiss firm A.G., which also supplies the ozone generator. No injury to health has been experienced so far in baths operated with ozone reconditioning. An essential part of the "Complex ozone-per mutate-mixed-bed filter" process is formed by the filter, inoculated with ions of heavy metals and metal oxides. The name of the "Indirect Quant ozone" process applies the use of ozone mixed indirectly into water. It is used particularly for the purification of the water in salt thermal baths. The "Combined Ospa Chlorine-Oxygen" process is based on the production of chlorine by the process of electrolysis. The electrolytic allows a concentrated oxygen content in the bathing water, with the consequent presence of traces of ozone. The number of processes clearly shows the headway ozone has made in this special field. Apart from the uses of ozone, an essential feature of all these processes is the incorporation of water purification steps into the process. A more detailed discussion of this subject, however, would go beyond the parameters of this paper. The sometimes sketchy description of the multitude of applications shows the increasing utilization of ozone in a great variety of technical fields. From this review in can be inferred that the uses of ozone in certain areas is not yet fully competitive with other long established methods. Thus ozone represents no cure-all, but it has definite advantages in many cases making worth-while a search for its economic use. In addition to the applications described here, a great number of possibilities will develop in the future for which only laboratory experiments have so far been carried out.
CHEMICAL REACTIONS OF OZONE The chemical reactions of ozone are related to its molecular structure. One of the oxygen atoms can be detached relatively easily, yielding there-by nascent oxygen, and this makes ozone practically the strongest oxidizing agent. In other reactions, the whole of the ozone molecule can be added to a reagent. Ozone takes part in inorganic reactions as an exceptionally powerful oxidizing agent. This behaviour follows also from its high redox potential, exceeded only by that of fluorine. It oxidizes metals (with the exception of gold, platinum, iridium) to oxidize of the metals in there highest oxidation states, oxides to oxides of higher oxidation or to peroxides, sulphides to sulphates, carbon to carbon dioxide (even at normal temperatures), and ammonia in either the dry or gaseous liquid states, of dissolved in carbon tetrachloride, to ammonium nitrate. ozone does not react with ammonium salts. In the majority of oxidation reactions, it is reduced to molecular oxygen releasing only one oxygen atom. Ozone can react in three different ways with organic compounds, vis. (I) a common oxidation reaction takes place, (II) peroxide compounds are formed or (III) addition to double or triple bonds is brought about, with the formation of ozonides, in many cases only as intermediates. Oxidation, particularly at high ozone concentrations, can go as far as the formation of CO2 and H2O. Usual concentrations, however, cause the formation of substances containing more oxygen or less hydrogen from the original molecule.
INTERACTION BETWEEN OZONE AND VARIOUS SUBSTANCES For materials in contact with ozone two considerations apply. The first relates to the extent ozone reacts to the materials in question, the second being associated with the magnitude of the effect exerted by the material on ozone itself to promote or catalyze its decomposition. Groups of materials encountered in practice will be investigated below according to the two view points referred to above. As has been shown earlier, most metals are strongly oxidized by ozone. Corrosive action can be generally noted over 2 to 3 ppm., Particularly in the presence of moisture. Lacquering or other surface treatments applied for corrosion protection can be beneficial to a certain extent. Metals promote in most cases the decomposition of ozone, some of the reactions being catalytic. Good catalysts are, for example, iron, particularly if rusted, zinc, mercury, platinum and silver. According to Yemelyanova et al, noble metals are the most active catalysts at low temperatures. Kastanov et al, found that, all of the metals, pure aluminium has the smallest catalyzing effect at low ozone concentrations and 373k. Schumacher found aluminium acceptable at low temperatures even for the stability of liquid oxygen-ozone mixtures, whereas, according to Lamneck decomposition is slower under such conditions, for example, in the presence of copper than in contact with aluminums Mahieux's investigations showed that a room temperature and an ozone concentration of 7%, decomposition of O3 is not assisted by pure lead, copper, or tin in addition to aluminums. Stainless steel, besides anodized aluminium is suggested as a metallic material for construction, particularly for applications involving low temperatures and high ozone concentrations. In terms of chemical resistance and stability of ozone, glass would be an ideal material for vessels and piping. Owing to its low strength and lack of elasticity, however, the use of glass is severely limited. For higher mechanical loads (due to pressure for example) and increased ozone concentrations therefore, the use of glass lined steel is being proposed. It is noteworthy that Waller and McTurk found that bottles made of stainless steel with the inside surface phosphate to be as satisfactory as glass. Minimum wall effect and maximum half-life for the thermal decomposition of ozone were experienced with such bottles. Due to their large specific surfaces, all absorbents such as activated carbon, molecular sleeves, silica gel, activated aluminums, etc., act as strong catalysts to assist the decomposition of ozone at room temperature. In the case of activated carbon, oxidation also takes place, whereas the pure absorption effect prevails in molecular sleeves because the catalytic effect ceases on saturation. The catalytic effect of molecular sleeves for preventing the decomposition of ozone can be well utilized in practice, e.g. for the de Ozonization of tail gases containing ozone. Judging from the investigations of Szabo a properly sized stainless steel reactor packed with 13X type molecular sleeves, is suitable for continuous de Ozonization at room temperature, yielding an ozone free outlet stream even with ozone concentrations in the inlet stream at the percent range. Due to the exothermic decomposition the part of the catalyst bed, the state of which is dependent on ozone concentration and flow velocity will be heated to a temperature above ambient. Melted pure X-Al2O3 containing relatively large pores has practically no catalytic effect on decomposition. The presence of a small amount of metallic oxide inclusions, however, can initiate explosive decomposition. Oxides of iron, cobalt, nickel, silver and manganese are particularly active catalysts. Pure (CU O), in turn, has practically no effect on ozone. Activity of catalysts is strongly dependent on their crystalline structure, the presence and distribution of moisture and other factors. A good catalyst for cracking ozone is soda lime, which is also used in practice together with metallic oxides and the absorbents referred to above for the removal of ozone from tail gases discharged into the atmosphere. Rubber, which is used in practice as material for seals, pipes and other components, like organic materials in general, reacts actively with ozone. Synthetic rubbers have a superior resistance to ozone over natural grades. From among plastics, fluorinated plastics can be used with advantage as materials for seals, pipes and even general construction materials, due to their resistance to the action of ozone and to their lack of catalytic action to promote decomposition. For high ozone concentrations, use of PTFE and polydichlorodifluorethylene is recommended. These plastics can be utilized for coatings, e.g. for lining steel bottles used in the storage of concentrated ozone.
BACTERICIDAL, STERILIZING AND OTHER EFFECTS IN LOWER ORGANISMS The use of ozone for decanted in water purification was mainly due to its toxic effects on micro-organisms found in water, effects which exceed those of any other disinfectant. Experience has demonstrated that it destroys with extreme efficiency the spores of molds, amoebae, and viruses, and bacteria as well as various pathogenic and saprophytic germs. These micro-organisms represent a wide variety of species, genera and families. Therefore, organisms were to be selected for further investigations which would best represent typical pathogenic effects on humans and animals. It was similarly the long and successful use of ozone that created interest in its utilization as a general germicide and sterilizing agent and for highlighting its advantages over the other germicides used generally for water purification, primarily chlorine. Special importance can be attached to investigations relating to the specific destructive power of ozone on selected bacteria, including quantitative data and the mechanism of sterilizing and germicidal effects. As is known, bacteria are microscopically small, single-cell creatures having a primitive structure, and they take up foodstuffs from and release metabolic products to the exterior and multiply by division. The bacteria body is sealed off towards the exterior by a relatively solid cell membrane. Their vital processes are controlled by a complex enzymatic system to which macro-molecular organic compounds, frequently containing phosphorus or sulphur, contribute. Viruses are extremely small, independent particles, built up by crystals and macromolecules. Unlike bacteria they multiply only within the host cell. They transform the protein of the host cell, to a certain extent autocatalytic ally, in proteins of their own. Germicides and sterilizing agents interfere with the metabolism of bacterium-cells, most likely through inhibiting of blocking the operation of the enzymatic control system. A sufficient amount of oxidizing agent breaks through the cell membrane and this leads to the destruction of the bacteria or virus. The free electrical charge of the cell membrane constitutes in most cases a strong obstacle for the effective operation of the disinfectant. Chlorine is known to enter into reaction with water and the reaction products generated will have a distribution depending on the pH of the water. Free chlorine and un dissociated HOCI can penetrate relatively easily into the bacterium cell; penetration , however, is not so easy for the negative OCI (Hypo chlorite) ion. Therefore, it is more difficult for chlorine to kill germs in an alkaline solution (pH > 7) where dissociated OCI ions are in preponderance. The destruction rate of germs depends in general, on the concentration, the number of bacteria in unit volume and on the pH of the medium. The process of necrosis of bacterium cells and the contribution of the penetration of germicides throughout the cell membrane and the part played by the various reactions taking place in metabolism have not been fully explored yet. On the basis of considerations referred to above, and Bering in mind that ozone does not react with water, it can be assumed that the free electrical charge of the cells does not reduce the effect. Holluta and Unger showed that the destruction rate of germs has no measurable dependence on pH. This fact constitutes one of the major advantages of ozone over other disinfectants. For comparison, the germicidal effect of ozone and chlorine on the basis of experiments conducted by independent investigators is show in table 21. For the experiments, the bacterium Escherichia coli was selected as a characteristic indicator of contaminants stemming from feces found in natural water. Despite the different circumstances, the better germicidal effect of ozone can be inferred from the comparison. Kizhinov and Kozhinov report, the bacterial action of ozone on the basis of measurements carried out at one of the municipal water works in Moscow. Measurements carried out by Fetner and Ingols indicated the need for higher ozone concentrations and longer exposure times under approximately identical conditions. Deviations can probably be contributed to changes in the technique used for the analyzes and to differences between experimental conditions. The curve for chlorine is logarithmic, however, the effect of ozone below a certain critical concentration value is small, of zero, but practically all germs above this level are destroyed. This effect is called all or none response and the critical concentration is referred to as "flash point". The critical concentration lies just at the level, generally between 0.4 and 0.5 mg dm that produce a small amount of residual ozone in water. The threshold value of 0.4 to 0.5 mg dm -3 was observed by several research workers in case of the viruses of influenza and polio, certain coli form bacteria and the spores of Clostridium botulinum. For water purification it is the residual concentration which is the critical factor in controlling the destruction of micro-organisms. At the beginning of the 1960's it was unanimously established that ozone solutions, particularly in the presence of free ozone, have a more rapid effect on viruses than attained by the action of chlorine. Thus, viruses prove to be resistant to chlorine under certain circumstances but, it is increasingly difficult to destroy them even by exposure to ozone. In general, the various genera of the polio virus was used for the experiments. Katzenelson et al put greater emphasis on the effect exerted on viruses during their investigations since they are known to be more resistant to disinfectants than bacteria. A noteworthy phenomenon of their investigation was the two step process of inactivation. Period 1 lasts less than 10 seconds at which time a kill rate of 99% is achieved. Period 2 runs for several minutes to complete destruction. Ozone was applied at 7 intermediate levels between 0.07 and 2.5 mg dm -3 but the phenomenon was independent of the changes in the concentration. According to Berg et al the higher resistance of viruses is caused by the formation of clumps. To establish the theory, a preparation of polio virus was subjected to ultrasound of 100w for 2 minutes at 20mhz. Ultrasound caused the breakdown of such virus clumps, which then became extremely susceptible to ozone. The other interesting observation was that the susceptibility of the virus to ozone exposure persisted for a long time, even after protracted storage of the culture at 203K. The virus relocated to a storage temperature of 258K, however, became resistant. To account for this phenomenon the most acceptable explanation appears to be that a significant percentage of the viruses form clumps. Sommerville and Rempel published data (referring to results obtained in 1943 by Kessel et al according to which the virus was inactivated within 1.5 to 3 hours at a residual concentration of 1 mg dm. At the same time, Naumann stated in 1954 that a residual ozone content of 0.45 mg dm inactivated the polio virus within 2 minutes. As virus transmission can take place in water, possibilities of inactivation had to be more precisely determined to prevent infection from viruses. In France Coin et al, investigated the inactivation of the poliomyelitis virus by ozone. They attained a kill rate of 99.99% with residual ozone content of 0.3 to 0.4 mg dm in water within 3 to 4 minutes. Gevaudan et al studied, under somewhat different conditions and by various methods, the destruction of viruses from the sabin species of the poliomyelitis III genus. They found that organic and inorganic substances in water reduce the effects of ozone. In other respect their findings were identical to those published by Coin et al in 1964. American investigators studied a variety of typical micro-organisms to determining the specific effects of ozone. These included Bacillus Anthracites (which causes Anthrax in sheep, cattle, and pigs, but is also a human pathogen), Clostridium Botulinum (its toxin paralyzes the central nervous system, being a poison multiplying in food and meals), Influenza virus, Bacillus Subtilis (hay Bacillus, it decomposes organic matter in soil and water, but not pathogens). For the experiments an average ozone concentration between 100 and 200 ppm. was used. Miller et al achieved a full sterilization of the spores of Bacillus Subtilis by exposing them to 100 ppm. of ozone for 45 minutes. Mice were inoculated with type A toxin with the bacterium causing botulism and cultures of egg with influenza virus. The culture contained a maximum of 10 bacilli causing botulism which represents the exact lethal dose for mice. An exposure of the culture for 30 minutes to ozone was generally sufficient for inoculated animals to survive. In the case of the influenza virus the egg cultures remained negative during the check examination. It was shown also for other micro-organisms that they could be destroyed by the application of 1.5 to 2.0 mg dm -3 ozone. Included in this class were the Klebs-Loffler Bacillus or Typhus Abdominalis, that spreads typically by aqueous infection and causes Typhoid. This group also contains the typical Staphylococci causing general inflammation as well as the spores of niger known under the name of Black Mount. At present, treatment at a suitable temperature is used for sterilization, but sterilizing by chemical means was also suggested. Results obtained from a large number of experiments carried out with water show that most probably ozone is the only substance which can be used as a chemical sterilizing agent to substitute for the effects of temperature. Ozone excites intense light emission of Armillaria mellea, a luminescent basidiomycete, over the concentration range of 75 to 500 p.p.m. investigated. Exposures running for 3 hours were not fatal for all of its species (269). This property favouring ozone is caused by a pigment of melamine type because another species of luminescent basidiomycetes have no such pigment was destroyed after an exposure to 100 ppm. of ozone for 10 minutes. For the progeny of Vicia faba, ozone causes chromosome aberration and its effect is twice that observed by x-rays.