Some biophysical principles underlying the controlled atmosphere storage of plant material

Transcription

1 Ann. appl. Biol. (I974), 78, Printed in Great Britain I49 Some biophysical principles underlying the controlled atmosphere storage of plant material BY W. G. BURTON Agricultural Research Council Food Research Institute, Colney Lane, Norwich (Accepted 4 April 1974) SUMMARY Controlled atmosphere (CA) storage is of use for commodities which potentially can undergo rapid and unacceptable biochemical change. In air, the oxygen status of most plant material, including fleshy storage organs and fruits, suffices, even in the centre, for cytochrome oxidase to be fully saturated. Conflict of evidence exists as to possible 0, and CO, gradients in fruit which, though physiologically unimportant in air, could be important under CA conditions. CA storage gives possible control of internal 0, from o to about 80-95%; internal CO, from about 3-4 to IOO yo ; and both simultaneously to intermediate values. Calculated molarities of dissolved 0,, CO, and ethylene are given for various atmospheres. The differences in the 0, concentrations recommended for different varieties of apple are not readily explicable. Varietal differences in susceptibility to CO, injury could possibly result from anatomical, rather than biochemical, differences. This could be determined partly by resolving the conflict of evidence mentioned above. Variability of plant material prevents precise control of intercellular atmosphere ; recommended atmospheres can be designed only to avoid completely anaerobic conditions and a harmful level of CO, in the centre of the least permeable individual fruit or vegetable. Effects of low 0, and high CO, are briefly described. INTRODUCTION This paper is concerned with the principles of controlled atmosphere storage, not with its practice, and more specifically with the influence of the ambient atmosphere upon the oxygen and carbon dioxide status of the cells and some possible effects of this upon metabolism. For a survey of the controlled atmospheres which have been recommended for the storage of a wide range of produce, reference should be made to Stoll (1973~); and to Fidler & Mann (1972) for details of the practical application of such atmospheres to the storage of pome fruits. If the useful storage life of a commodity is limited by biochemical change - as may be the case mainly in material which is in a state of, or has the potential for, rapid development or change when it is stored-then reducing the rate of change will increase the storage life. Chemical changes are subject to influence by temperature and by other factors, such as the concentration of reactants and accumulation of products. By controlling these we can influence the rate and direction of the reactions

2 I 50 W. G. BURTON in simple chemical equilibria and determine the final concentrations of the participating compounds. Translated into the environment of the plant cell, however, though the potential scope for control exists, the dynamic equilibrium, resulting from the many hundreds of inter-linked reactions which are proceeding, is so complex that we can rarely be certain of the effect of changing any variable. Nevertheless, as a first approach to the problem of controlling undesirable metabolic change we can reasonably make use of general principles which have been derived from observations on simple reactions and equilibria in vitro - for example, lowering the temperature decreases the velocity constant of a reaction; decreasing the concentration of reactants decreases the velocity at constant temperature; and accumulation of the products of a reversible reaction progressively decreases the net rate of change, until a state of dynamic equilibrium is reached in which this is zero. The two last were the rationale of the earliest experiments on controlled atmosphere storage. Respiration is an index of the rate of metabolic turnover, and reducing the rate of respiration could be considered as a possible means of lowering metabolic rate. Application of the Law of Mass Action to the simple concept of respiration being the oxidation of a sugar, through at least partly reversible steps, to give carbon dioxide and water, suggested that if the carbon dioxide in the storage atmosphere was augmented, and the oxygen reduced, respiration might be reduced and storage life extended. In fact both a reduction in the rate of respiration and a correlated extension of storage life were obtained (Kidd & West, 1927a, 6; Kidd, West & Kidd, 1927). It was well understood by these early workers that the physiological basis of their work, as just stated, was probably an over-simplification (Kidd et al. 1927), for instance, we now know that respiration and other processes occurring during the ripening of fruit can be influenced separately (Dostal & Leopold, 1967; Frenkel, Klein & Dilley, 1968; Rhodes, 1970); but in the state of knowledge at the time, Kidd & West made a logical and reasonable jump into the dark and landed on a commercially successful method of storage, certainly of pome fruit, which has stood the test of time for nearly fifty years. In the early applications the increase in carbon dioxide and depletion of oxygen, achieved by storing the respiring fruit in a gas-tight chamber with a controlled air leak, were approximately equal, and the concentrations of each were around 10%. It was recognized at the very beginning, however, that separate control of the oxygen depletion and carbon dioxide increase would be preferable (Kidd et al. 1927) and recommendations for the storage of some varieties of apple in low oxygen and comparatively low carbon dioxide (e.g. 2.5 % O,, 5 yo CO,) were made by Kidd & West (1935). Storage in low oxygen with low carbon dioxide is now common practice in the case of commodities and varieties for which it has been found desirable. In Table I are given the combinations of temperature and atmosphere which have been recommended for the storage of some commodities. THE DYNAMIC EQUILIBRIA BETWEEN PLANT MATERIAL AND THE ATMOSPHERE The major part of the respiratory activity of living plant material is centred in the mitochondria, in which the cytochrome system is localized; though enzymes other than cytochrome oxidases (e.g. ascorbic oxidase and phenolases), with a lower

3 Biophysical principles of controlled atmosphere storage 151 affinity for oxygen, and not located in the mitochondria, will combine with oxygen if this is present in sufficient concentration. The components of the overall dynamic physical equilibrium with respect to oxygen may be represented by: (I) The combination of any oxidases with a sufficiently great affinity for oxygen, relative to the concentration dissolved in the cell sap, with this dissolved oxygen, thus reducing the concentration in solution below that in equilibrium with the partial pressure in the gas phase in the intercellular space. Apple Table I. Conditions which have been recommended for the controlled atmosphere storage of various commodities Commodity Temperature ( C) Blenheim Orange 4 5 Bramley s Seedling (UK) 3-4 Cox s Orange Pippin (UK) (to Feb.) (beyond Feb.) Cox s Orange Pippin (Swiss) 4 Golden Delicious (Swiss) 2.5 or : 2.5 Jonathan (UK) 4 5 Jonathan (Swiss) 4 King Edward VII (UK) Pear Conference (UK) Conference (Swiss) 0 Doyenne du Cornice (UK) 00.5 Doyenne du Cornice (Swiss) 0 William s Bon Chretien (UK) William s Bon Chretien (Swiss) 0 Atmosphere (%) P CO, 0 2 Air only 8-10 c * 5 < I c * IS* 2 2 Blackcurrant Cauliflower (Swiss) Cucumber (Swiss) I4 5 5 Cabbage (Swiss) Red Savoy White * No scrubber. C02 controlled. 02+C02 assumed to total 21%. Reference Fidler & Mann (1972) Fidler & Mann (1972) Fidler & Mann (1972) Fidler & Mann (1972) StOll (1972) St011 (1972) StOll(I972) Fidler & Mann (1972) St011 (1972) Fidler & Mann (1972) (2) A movement towards re-establishing equilibrium between the gas phase in the intercellular space and the oxygen in solution by passage of oxygen from the former to the latter, thus reducing the partial pressure in the space relative to that in the atmosphere surrounding the fruit or vegetable. (3) A movement towards re-establishing equilibrium between the partial pressure in the intercellular space and the ambient atmosphere by diffusion of oxygen from the latter to the former. This system includes definite barriers to the free establishment of equilibrium. If we take the steps in reverse order to the above, thus following the direction of movement of the oxygen, this must first diffuse in the gas phase from the atmosphere into

4 W. G. BURTON the intercellular space through pores in the outer integument of the fruit or vegetable; it must then dissolve in the water permeating the wet cell walls surrounding the intercellular space, diffuse in solution from the outer surface to the inner surface of the cell wall, and afterwards diffuse to the enzyme with which it combines, through potential barriers in the form of cytoplasmic and mitochondria1 membranes. The above represents the simplest system, in which the anatomy of the material is presumed to offer no internal physical barriers to diffusion, other than those at single cell level. In fact, these other internal physical barriers may occur, there being some evidence, discussed later, that the vascular region of the apple fruit may present such a barrier (Brandle, 1968). Burton (1950) studied aeration and equilibration with oxygen in the potato tuber, in which the volume of the intercellular space is only about I-2% of the volume of the tissue (Gorter & Nadort, 1941; Burton, 1950; Burton & Spragg, 1950). He measured the oxygen in solution in the cell sap and found it to amount, on average, over the range 5-25 "C, to 94% of that which would have been in equilibrium with the concentration in the gas phase in the intercellular space, also measured. In the potato therefore the barrier to oxygen movement to the respiratory centres represented by its entering into solution in the cell sap, from the gas phase in the intercellular space, Table 2. Concentrations of dissolved oxygen in potato sap calculated to be in equilibrium with different concentrations in the gas phase" '2, Oa in gas phase... I 5 10 I5 20 Temp ("C) r Equilibrium conc. in sap (M x IO-~) 0 1' ' '3 10 1'5 7' I5 1 ' '5 20' ' ' ' I * On the assumption that the solubility of oxygen in the sap is about the same as the solubility in a 0.4 M sucrose solution, behaving similarly to nitrogen in this respect (see Burton & Spragg, 1950). would appear to be slight. The comparatively small effect upon oxygen uptake, of changing intercellular oxygen concentrations over a range of about 3-98 yo (Mapson & Burton, 1962; see also Craft, 1963), and indeed, the absence of any effect in the middle of the storage season (Burton, 1974) would suggest that the other barriers to the diffusion of oxygen in solution, within a single cell, are not serious; and that in fact the cytochrome-c oxidase system in the mitochondria is fully saturated at normal intercellular oxygen concentrations, which range from averages of about I 3 yo at 25 "C to about 18% at 5-10 "C (Burton, 1950). In all other plant tissues, which respire at rates of the same order of magnitude as the potato, a similar situation will hold, provided that each cell is in contact with the intercellular space, and that this is continuous and interconnected throughout the tissue. If there were a break in continuity, or if each cell were not in contact with the space, but had to depend upon diffusion over some distance in solution, then the picture would be very different. In potatoes respiring uniformly at 2 ml kg-l h-l, cells more than about 5 mm from an intercellular space, containing oxygen at a tension of 0.18 atm, would be anaerobic (see A

5 Biophysical principles of controlled atmosphere storage I53 Gerard, 1931; Goddard, 1947; Burton, 1950). In the case of an apple respiring, at the climacteric, at, say, 10 ml kg-l h-l, the distance would be reduced to 3 mm, and in material respiring at as much as roo ml kg-1 h-l would be I mm. In Table 2 are given the concentrations of dissolved oxygen in potato sap calculated to be in equilibrium with different concentrations in the gas phase. There is no reason to think that the concentrations in other plant sap would differ markedly from these figures. THE INTERCELLULAR SPACE AND ITS ATMOSPHERE The efficiency of the intercellular space as a means of aerating the tissue depends upon its volume, its continuity, the degree to which it is filled with gas or is injected with liquid, and the levels of oxygen demand and carbon dioxide output with which it must deal. In most tissues with which we are concerned-exceptions being, for example, the lysigenous oil cavities of citrus fruits - the individual spaces are formed by the splitting apart of cells through the middle lamella, and increase in size as adjacent cells tend to round off during enlargement. This produces a continuous or almost continuous system, as can be demonstrated by injection with coloured liquids. Its volume varies very considerably - in leaves of different plants the extreme limits of the published figures range from 3-5 to 66% of the volume of the leaf, although most are of the order of 20-30% (Spector, 1956). Volumes reported for fleshy organs vary from less than I % in some potatoes (Burton, 1950) to about 36% in Lord Derby apples (Smith, 1938~). The space is normally filled with gas, even in apparently wet material such as the flesh of soft fruit, exceptions again being spaces such as the oil cavities mentioned above (see e.g. Eames & MacDaniels, 1947). The spaces in senescent material, too, may become injected because of membrane breakdown; an effect which, even if it is local at first, can be autocatalytic as a result of the anaerobic conditions induced. A good example is provided by the results of Kidd & West (19496) on the internal atmosphere, and condition of the fruit, during the storage of Conference pears in air at 12 "C, over a period of 50 days. The efficiency of aeration provided by the normal gas-filled intercellular space has been considered in detail by Gerard (1931), Goddard (1947) and Burton (1950). In leaves, the fact that photosynthesis occurs rapidly in a low ambient CO, concentration indicates that the tissue is very porous, but more direct estimates of porosity can be obtained by a combination of porometer readings and measurements of the stomata (Strebeyko, 1965). Even with the very small space in the potato tuber, Burton (1950) calculated that the fall in oxygen concentration from periphery to centre of a spherical tuber - positively related to the rate of oxygen consumption and to tuber size -would be unlikely to exceed about br2/6 %, where 6 was the rate of oxygen uptake in ml ml-l tissue h-' and r the radius in mm. This indicates that at normal rates of respiration the gradient of oxygen in a tuber would probably not exceed about 0.5%. In no tissue which is considered in this paper, in which rates of respiration, volumes of intercellular space, and composition of the gas in this, have been determined, have the values been such that, under normal storage conditions in air, the gradient of oxygen, in uniform tissue, would be likely to be of much physiological significance. The banana must, however, be specifically excluded from this generalization (Wardlaw &

6 I 54 W. G. BURTON Leonard, 1936, 1939; Brandle, 1968). Smith (1947) found gradients of yo in the flesh of apple fruits over a radial distance approaching 2 cm. The lowest concentraticn of oxygen he recorded, for fruit stored in air at 12 "C, was 16.6% - the resistance of the peel and not of the flesh being the reason for the major part of this drop below the ambient level. If we are considering the uptake of oxygen by cytochrome-c oxidase with its very high affinity for oxygen (for example published values for KnL, with respect to oxygen, for the high-affinity terminal oxidase of the potato tuber vary from 3 x 10-6 M to 7 x IO-~ M; see Burton, 1974) then a fall to 16.6% is insignificant - and indeed a fall to I yo would not be important. Brandle (1968) attempted to measure the degree of saturation with oxygen at various points in apples, potatoes and bananas held at 20, using a miniature Clark electrode. Superficially, the values for oxygen status he obtained for the potato would be consistent with those of Burton (1950), and for the apple might not be incompatible with those of Smith (1947), allowing for the higher temperature and an oxygen uptake in excess of 8 ml kg-l h-l. The gradient which he found in oxygen status in the apple, from periphery to centre, would be impossible, however, on the assumption of an interconnected and completely gas-filled intercellular space. There was a fall of thc order of 5% between the periphery and the ring of vascular bundles. In this region there was a sudden steepening of the gradient, followed, still further towards the centre, by a less steep fall. The results would be consistent with there being a vascular zone, 1-5 mm in thickness, in which the intercellular spaces were either so small or infrequent or so infrequently connected, that it represented a barrier at least equal to that presented by the outer integument, Both inside and outside this vascular zone the tissue appeared to be more porous, but the comparative impermeability of the vascular region led to a minimum concentration of oxygen within it, just outside the horny core lining of the seed cavities, in equilibrium with between about 3 and 63; in the gas phase. In the apple fruit, we may thus have two zones - an outer one in which at usual storage temperatures and pre-climacteric rates of respiration we might expect an intercellular atmosphere in the range of about 17-19% 0, and 2-47; CO,, and a zone inside the vascular ring in which the intercellular concentrations lie in the range of perhaps 12-15% 0, and 6-9% CO, (it must be remembered that Brandle's 3-6s 0, was at 20 "C). Again, considered in relation to the oxygen affinity of cytochrome-c oxidase, the fall below the oxygen concentration in air is physiologically unimportant. The above figures for oxygen concentration, and those used in later discussion, have deliberately been based upon the results of Brandle (1968), which present the least favourable picture of the porosity of apple tissue. Other workers have obtained very different results suggesting a much more porous flesh (e.g. Smith, 1947; Hardy, 1949; Hulme, 1951; Burg & Burg, 1965) with much smaller concentration gradients and with no suggestion of an increased gradient in the vascular region, It would appear desirable to re-examine critically the oxygen status and intercellular atmosphere of fleshy fruits, coupled with measurements of the resistance of the outer integument to gaseous diffusion by some such method as that of Burton (1965), involving no mutilation of the tissue. It should be noted that Brandle's method involved measuring the oxygen status of what he believed to be small pockets of sap at the base of an

7 Biophysical principles of controlled atmosphere storage I55 incision by a hypodermic needle. In such pockets of mutilated tissue oxygen uptake would be potentially great and diffusion of oxygen would be in solution over distances of perhaps mm. This would explain an erroneously low value for oxygen status - see the end of the section on ' The dynamic equilibria between plant material and the atmosphere' - but does not readily explain a steep gradient from periphery to centre, in that the effects of mutilation could be thought to be similar at every depth. In the potato tuber, and, according to the results of most workers, in the apple and pear, the most important factor influencing the composition of the atmosphere of the intercellular space is the resistance to diffusion provided by the outer integument, coupled with the metabolic oxygen demand and carbon dioxide output of the tissue. The resistance can be expressed most simply for our present purpose in terms of the rate of diffusion of oxygen or carbon dioxide activated by, say, a I yo deficit or surplus, respectively, in the intercellular space immediately under the integument. The rate is a function of the surface area of the commodity and really should be expressed as such; but it is possible, for any one commodity, without great inaccuracy, to relate intercellular deficit or surplus to uptake or output expressed in the practically more meaningful ml kg-1 h-l, on the grounds that for any given commodity, of marketable size, the surfacelweight ratio does not vary widely. In Table 3 are given a number of such relationships. Given some knowledge of the minimum and maximum gradients which are likely to be encountered in the flesh, these figures give us a possibility of deducing the internal atmosphere at any rate of respiration in any atmosphere. Table 3. Rates of di#usion through the integument, and internal atmospheres immediately under the integument, of some samples of apple, pear and potato Internal Diffusion atmosphere (%) through skin immediately ml kg-' h-l/x % under skin gradient && Reference Commodity co2 0 2 co2 0 2 Apple (freshly harvested) 12 "C Cox's Orange Pippin 3' '3 1.8" Kidd &West (1949~) King Edward VII '9 I.8 I.5* Kidd & West (1949~) Stunner Pippin '7 3.3* Kidd & West (I 949 a) Pear (5 days after harvest) 12 "C Conference '4 2'I* Kidd &West (19496) Potato (stored) Arran Consul, 10 "C 1.8 i8.2 I '0 0.7~ Burton (1950) Arran Consul, 25 "C 7'0 I * Burton (1950) Majestic, 10 "C 2'2 I 8.3 1'1 0.9 Burton (1974) * Derived from C02 output on the assumption that RQ = I. OXYGEN AND CARBON DIOXIDE EQUILIBRIA IN CONTROLLED ATMOSPHERES Let us now consider the effect of controlled atmosphere storage upon the internal atmosphere, taking two typical atmospheres as examples - 11 yo 0,, 10% CO, in a store with no scrubber; and 3 yo O,, 5 yo CO, in a scrubber store - and taking the

8 ~ 156 W. G. BURTON fruit, respiring at 4 ml kg-' h-l in air, to be placed immediately in them. Normally, with gradual atmospheric change, the initial response given below would be absent. In the first of these atmospheres, the immediate response would be diffusion of carbon dioxide into instead of out of the commodity, and diffusion of oxygen out; both in response to reversed concentration gradients, and in most cases, because of the size of these reversed gradients, both at rates which initially would be multiples of those of the previous diffusion in the reverse directions as a result of respiration. Over a period of hours, however, a new state of equilibrium would be reached in which, assuming the biochemical combination of oxygen and production of carbon dioxide to be the same as in air (but see below), the percentage of carbon dioxide in the intercellular atmosphere would be 10% more, and that of oxygen 10% less than in air. For the fruit listed in Table 3, but respiring at 4 ml kg-l h-l, this would be in the range S-9% 0, and 11-12% CO,, immediately under the skin, the gradients of oxygen and carbon dioxide having been re-established at the slopes previously existing in air, necessary to activate diffusion equal to the unchanged uptake and output. Taking Brandle's picture of the oxygen status throughout the flesh of the apple, we might expect oxygen concentration in the flesh to lie within a range of about 6-9% outside the vascular region and about 2-4% within it. If we assume a similar distribution of carbon dioxide, this would be about 11-14% in the outer zone and 16-18Y/, Table 4. Concentrations of dissolved carbon dioxide free in solution in potato sap calculated to be in equilibrium with diflerent concentrations in the gas phase" 3, CO, in gas phase... I 5 I Equilibrium conc. in sap (M x IO-~) Temp ("C) ' '5 5 5' ' I 14.0 I '7 71'5 '15'5 '5 4' ' " '2 * On the assumption that the solubility of carbon dioxide in the sap is about the same as the solubility in a 0.4 M sucrose solution, behaving similarly to nitrogen in this respect (see Burton & Spragg, IC~~O). in the inner zone. There is no physical reason why respiration in the controlled atmosphere should differ from that in air, because it has been possible for all the gradients to be re-established. If we consider biochemical reasons, the level of oxygen is such that uptake by the cytochrome system would be practically unaffected, though admittedly any uptake by low affinity oxidases could be considerably reduced - for example the K,,, of phenolase, with respect to oxygen, is in the region of x IO-~ M (Mapson & Burton, 1962). On the other hand the carbon dioxide will have been increased some two- to four-fold in the gas phase, with a proportionate increase in dissolved carbon dioxide (Table 4). There will be a corresponding tendency for the back reaction to occur in reversible decarboxylation processes, and consequent re-establishment of concentration equilibria in these and linked reactions, which could in turn affect oxygen demand. In fact Fidler & North (1967) found that in similar gas mixtures there was a 60% decrease in CO, output and also a 40% decrease in 0, uptake. If the carbon dioxide in the atmosphere were kept at 0% and oxygen

9 Biophysical principles of controlled atmosphere storage I57 reduced to 10 yo then there was a 20 % decrease in uptake. This last would result from a reduction in uptake by enzymes, such as phenolase, with a comparatively low affinity for oxygen. On the basis of Fidler & North's figures the internal atmosphere CO, outside the vascular ring would be established at about 8-10% 0, and I 1-12y~ and about j-67i 0, and 13-14% CO, within it. Though the oxygen is halved in comparison to storage in air, the oxygen status remains high, at least as far as the main respiratory enzyme is concerned, though uptake by enzymes such as phenolase would be much reduced. The carbon dioxide has been doubled. Now consider the second atmosphere ,, j yo CO,. The reasoning is just the same, and need not be repeated in detail. Fidler & North (1967) found, for this atmosphere, a 60% reduction in oxygen uptake and a 68% reduction in carbon dioxide output. Taking this into account we would expect the internal atmosphere to be established at about 1-2% 0, and 6-7% CO, in the outer zone and less than I yo 0, and about 8-9% CO, in the inner zone. In such an atmosphere the internal carbon dioxide concentration, because of reduced output, is perhaps on average about one and a half times that in air, though in the centre it may only be about the same as in air. The oxygen status, as far as cytochrome-c oxidase is concerned, is still adequate outside the vascular ring; though inside this it is less than adequate for full functioning. There would be no possibility of oxygen uptake by low-affinity enzymes such as phenolase. Whatever the oxygen affinity of the enzyme systems, however, there is no possibility of oxygen uptake exceeding that which could be supplied by diffusion activated by the difference between the ambient concentration and a nearly anaerobic intercellular space - i.e. nearly 3 %. Thus if the potential uptake increased, as during the climacteric, if the storage temperature permitted this to occur, there is no physical possibility of satisfying a demand greater than, in the case of the examples in Table 3, between rather less than 4-5 ml kg-l h-' in the King Edward VII apples and rather less than 9.9 ml kg-' h-l in the Sturmer Pippins. The controlled atmosphere recommended for prolonged storage of Cox's Orange Pippin in Table I ( % 0,, < I yo CO,), if we assume an integument permeability as in Table 3, offers no possibility of an oxygen uptake in excess of 3.3-4'5 ml kg-1 h-l. This atmosphere also would result in less dissolved carbon dioxide in the flesh than in airstored apples, despite the ambient level being higher than in air, because of an approximately halved output (see Fidler & North, 1967). We might expect a maximum of between 3 and 6% CO, inside the vascular region. POSSIBILITIES AND LIMITATIONS OF CONTROLLED ATMOSPHERE STORAGE AS A MEANS OF INFLUENCING CELLULAR ENVIRONMENT It is obvious from the foregoing that controlled atmosphere storage is a means of varying the oxygen and carbon dioxide in the intercellular atmosphere quite considerably, but that there are limits to the variation imposed by the structure and respiration of the plant material, and the response of the respiration to variation in the atmosphere. The limits of variation will differ in different material but will be approximately as follows: Oxygen. Completely anaerobic conditions are readily achieved with no ambient

10 W. G. BURTON oxygen. The upper limit of internal oxygen is obtained in 1007~ 0, and varies with the rate of respiration achieved in this concentration. The uptake will tend to be maximal, although not necessarily much higher than in air, unless there is a potentially high uptake by low oxygen-affinity enzymes; and this uptake can lead to a steep gradient in the tissue. Ambient 100% 0, could well result in only 80% in the centre of a commodity such as the banana, the remaining 20% being CO,. In general we might expect of the order of % 0, in the centre of different commodities, rising to about 95-96% at the periphery, immediately under the integument. Carbon dioxide. The intercellular concentration of carbon dioxide is easily increased by increasing the ambient concentration, complete saturation being obtainable in 100% CO,. To decrease the concentration of carbon dioxide below that existing in air it is necessary to reduce the output while retaining a negligible ambient concentration. Reduction in the concentration of oxygen is a means of achieving this, though it may be necessary to reduce the oxygen to a very low level to obtain an appreciable effect. For example, in the potato in the middle of the storage season, when low-affinity oxygen uptake is absent (Burton, 1974), it would be necessary to reduce the internal oxygen concentration to about 0.5 % in order to halve the carbon dioxide output and concentration. This would necessitate an ambient concentration of 2% or less. In the apple, Fidler & North (1967) found a very similar reduction in ambient oxygen concentration to be necessary (3-2% 0, resulted in 63% of the CO, output in air; 1.5 yo 0, gave 39 yo of the output). It is thus possible to vary the carbon dioxide concentration in the tissue between perhaps half that which normally occurs in air and 100%. It should be noted, however, that a reduction in carbon dioxide concentration below that in air can only be achieved in low oxygen concentrations. It is not possible by controlled atmosphere storage alone to couple a low internal concentration of carbon dioxide with a high concentration of oxygen, because of the high output of carbon dioxide. It is, however, readily possible to couple a high internal concentration of carbon dioxide with a low concentration of oxygen; and simultaneously to increase the internal concentrations of both up to about 50% (theoretically attainable in about 60-70% 0,, 30-40q/o CO,). DIFFERENCES BETWEEN ATMOSPHERES RECOMMENDED AS OPTIMAL FOR DIFFERENT VARIETIES Table I shows that the controlled atmospheres recommended for the storage of apples may differ markedly for different varieties. Theoretically, this could be because the respiration and the permeability of the skin and tissue vary so greatly between varieties that in fact the environment of the cells is similar despite the very different ambient conditions. As to this possibility, Brandle (1968) found some difference between varieties in the gradients in oxygen status in the flesh, but calculation from his results would suggest that, at 4 "C, oxygen concentrations at the centre of an apple in air would not differ by more than about 2% between varieties. The difference would be less in controlled atmospheres because of reduced uptake. Kidd & West (1919a) found differences to be small between the peripheral internal

11 Biophysical principles of controlled atmosphere storage I59 atmospheres and rates of respiration of a number of varieties stored at 12 "C over a period of time. In particular, the average internal atmospheres of Cox's Orange Pippin and Blenheim Orange were almost identical, and their rates of respiration not dissimilar, indicating a very similar permeability of the skin to diffusion. Yet Cox's Orange Pippin, according to Fidler & Mann (1972) should be stored at "C with, for late storage, % 0, and I % CO,, whereas Blenheim Orange should be stored only in air at 4.5 "C. Under these conditions we can calculate, in the light of the previous discussion, that the sap of Cox's Orange Pippin contains 2-4 x IO-~ M CO, and 1-2 x IO-~ M 0,, whereas that of Blenheim Orange contains about 3-5 x IO-~ M CO, and x I O M ~ 0,. The difference in dissolved CO, is unimportant, while 1-2 x IO-~ M 0, would not normally be regarded as a harmfully low level in plant cells, and the difference between the recommendations is not readily explicable. It is still less explicable in the wider context of the survey of recommended conditions published by Stoll (1973~). These included 162 recommendations for the oxygen content of the atmosphere of scrubber stores, relating to fifty-six varieties of apple. In the great majority of cases the content recommended was 3 % or below - including 2% recommended by Kaess (1943), in Germany, for Blenheim. Only in eight cases did the recommendation exceed 4y0, and the highest content recommended was 7y0, for a variety (Sturmer Pippin) for which another recommendation was 3 yo. Varieties differ markedly in their susceptibility to carbon dioxide injury in the form of a brown discoloration of the flesh within the vascular region. Some, e.g. Bramley's Seedling and King Edward VII, will tolerate 8-10% CO, in the storage atmosphere in an unscrubbed store, conditions which could give, at 4 "C, about 6 x ~ CO, in the sap near the periphery, rising to about 8 x I O-~ M ( = 13 yo CO,) within the vascular region; together with about 1-7 x I O-~ M 0, near the periphery, falling to about 9 x IO-~ M. Other varieties, e.g. Newton Wonder, will tolerate only about 3 % CO, in an unscrubbed store, possibly giving about 3 x I O-~ M CO, near the periphery rising to about 5 x 10-3 M ( = 9% CO,) within the vascular region. Although a difference in tolerance of from 3 to 10% CO, appears quite large, the difference in dissolved CO, within the vascular region, making due allowance for reduced output, is proportionally quite small. Moreover, these figures for dissolved CO, are based on approximate estimates of the intercellular atmosphere within the vascular region. These estimates suffice when discussing large differences in oxygen status but are an inadequate basis for postulating a different biochemical tolerance of the cell to carbon dioxide when the proportionate difference in CO, status is so small. It could be argued that the limit of tolerance is about 6-8 x I O-~ M in every case and that varietal anatomical differences, particularly in the vascular region, could lead to this limit being reached at different ambient concentrations. Such a suggestion would involve differences no greater than that suggested by Brandle (1968) between the oxygen status of Oetwiler Orangenreinette (= 7.6% 0,) and that of Bohnapfel (= 3.6% 0,) both at 2 cm beneath the periphery, though at a high temperature (20 "C). Again it would appear that the conflict of evidence between Brandle and other workers as to gradients in the flesh should be resolved. Certainly, the localization of CO, injury would lend support to Brandle's concept of a comparatively impermeable vascular region.

12 I 60 W. G. BURTON ETHYLENE PRODUCTION AND ITS CONCENTRATION IN THE TISSUE All plant material produces volatile substances other than carbon dioxide - the characteristic smell of many commodities is sufficient indication of this. Such volatile substances may in many cases be minor metabolic by-products of no physiological importance, and the more refined our methods of analysis the greater the number which can be detected, for example, over IOO volatile substances have been detected in apples (Drawert, Heimann, Emberger & Tressl, 1968). Ethylene is in a different category, in that it is produced by ripening fruits in greater quantities than any other volatile except carbon dioxide, and is of great physiological importance as a ripening hormone. It is not produced at detectable levels by unripe fruit, but the start of production immediately precedes the climacteric rise in respiration and increases over a period of days to a rate, in Cox's Orange Pippin, of 5e60pl kg-l h-' at 12 "C (Meigh, Jones & Hulme, 1967). The post-climacteric rate of production is dependent on temperature (e.g. from 20pl kg-l h-' at 0" to 160yl kg-' h-' at 25" in the variety Laxton's Superb; Tomkins & Meigh, 1968) and on variety (e.g. at IS", from 20 p1 kg-l h-l in Newton Wonder to IOO pl kg-i h-l in Laxton's Superb; Tomkins & Meigh, 1968). The rate of diffusion of gases is inversely proportional to the square root of the density, and the rate of diffusion of ethylene thus approximates closely to that of oxygen under a similar concentration gradient. We can therefore use the figures for oxygen in Table 3 to calculate the concentration of ethylene in the peripheral intercellular space which corresponds to a given rate of production. These show that evolution from apples and pears at a rate of I pl kg-i h-l would be activated by a peripheral intercellular pressure, in the absence of ambient ethylene accumulation, averaging about 5 x I O-~ atm (i.e. c. 0-5 Pa). The solubility of ethylene in water ranges from about 2.3 x I O-~ ml ml-i Pa-I at 0" to about 1.2 x I O-~ ml ml-' Pa-' at 20, and we can probably assume a solubility in the aqueous component of plant sap of about 2 x IO-~ ml ml-l Pa-1 at 0" falling to about IO-~ ml ml-l Pa-' at 20, i.e. c. IO-~ M falling to j x IO-~M. Production at a rate of I pl kg-' h-' would, on this basis, be accompanied, in the absence of ambient accumulation, by a concentration in the sap, near the periphery, ranging from about 5 x IO-~M at 0" to 2.5 x IO-~M at 20, and about 4.3 x IO-~ M at a normal storage temperature, for apples, of 4 "C. Assuming uniform production throughout the tissue, and tissue porosity as suggested by Brandle (1968) we might expect a gradient, in tissue producing I pl kg-l h-l at 4", of from roughly 10-7 M at the centre to 4 x IO-~ M at the periphery, and proportionately more at higher rates of production - for example from 1-5 x IO-~ M to 6 x IO-~ M in postclimacteric Cox's Orange Pippins producing 15 pl kg-' h-' at 4". There would be more ethylene in fruit in controlled atmosphere stores in which ethylene had accumulated. At any given rate of production the effect of accumulation upon internal concentration is additive. For example, at a rate of production of 10 pl kg-l h-l, in the absence of accumulation, the intercellular tension might range from about 5 to 12 Pa, and the corresponding concentration in the sap at 4 "C from 4 x 10-7 to IO-~ w. In the presence of IOO ppm (= IO-~ atm; c. 10 Pa) accumulated ethylene, the intercellular tension would range from about 15 to 22 Pa and the

13 Biophysical principles of controlled atmosphere storage 161 concentration in the sap from about 1.2 to 1-8 x ro4 M. Ethylene in the ambient atmosphere can be physiologically active at concentrations of the order of I ppm (i.e. at a partial pressure of 10-1 Pa). In the absence of production or loss in the tissue this could be expected to lead to diffusion through the integument at a rate, diminishing with time, but initially about 0.2 pl kg h-l, until saturation in the sap was reached at a concentration ranging from about IO-~ M at oo to 5 x I O-~ M at 20, which can thus be deduced to be a physiologically active range of concentration. NECESSARY PRECISION OF CONTROL OF ATMOSPHERIC COMPOSITION It is essential, in making commercial recommendations, to state that precise control of atmospheric composition is necessary; otherwise great variability would be encountered in practice. Such recommendations should not be taken to mean that the precision recommended is always biochemically or biophysically necessary or attainable - in fact biological variation can be sufficient to cause the cellular environment to differ quite markedly even with very precise control, which thus gives a fallacious impression of the effective precision which is being attained. For example, Kidd & West (1949~) found the internal CO,, near the periphery of six individual Cox's Orange Pippin apples, to vary from 3.37 to 6.50% and the output of CO, from 6.2 to 8.0 ml CO, kg-l h-l, the two variables being uncorrelated. The peripheral intercellular oxygen varied from to 16.32%. If we placed these apples in 5% CO,, 3 % 0, and controlled the components of the atmosphere precisely within limits of fo-~y~, if this were possible, then, taking the CO, output and 0, intake to be reduced, respectively, to 32 and 40% of the values in air, as in the experiments of Fidler & North (1967), we might expect the internal CO, to vary from 6.0 to 7.2% and the internal 0, from o to 1.3 yo. These values would be near the periphery and superimposed upon them would be the increased range, at least of CO,, resulting from gradients in the flesh. The control is thus not as precise, at the point where it is effective, as superficially it appears to be. There thus appears to be little point in specifying atmospheres with a degree of precision which is unattainable intercellularly. Control to the nearest per cent is probably adequate, oxygen concentration being specified at a level sufficiently high to avoid completely anaerobic conditions in the centre of the commodity; and the carbon dioxide concentration sufficiently low to avoid carbon dioxide injury, as discussed above, also in the centre. These safe levels can only be determined by experience. EFFECTS OF LOW OXYGEN AND HIGH CARBON DIOXIDE IN THE TISSUE What we achieve by controlled atmosphere storage is achieved by its influence upon the substrates and activities of the enzymes involved in the changes we are trying to delay. In most cases our knowledge of the reactions involved is fragmentary, and little would be gained, in our present state of ignorance, by attempting to specify the biochemical nature of the effects. What is done below is merely to describe the influence of the concentrations of oxygen and carbon dioxide upon various characters relevant to quality. The list is not exhaustive.

14 I 62 W. G. BURTON Discolorations at cut surfaces are typically caused by the oxidation of phenolic substrates by phenolases, which, as we have seen, have a comparatively low affinity for oxygen, and are therefore susceptible to moderate changes in oxygen tension. Thus browning of the cut ends of broccoli (Smith, 19383) and lettuce (Singh, Yang & Salunkhe, 1972) is reduced or almost prevented by oxygen concentrations of 5% or below, as is the blackening of pre-peeled potato tubers. Loss of chlorophyll occurs during the ripening of fruit in storage and during the senescence of leafy material. It is much hastened by increased oxygen (see e.g. the results of Kidd & West, 1934 with 100% 0,). It is delayed by increased carbon dioxide, up to 15% (see e.g. Wang, Haard & Dimarco, 1971). Thus 10% CO, will retain the green colour of apples, though it cannot be employed if the variety is susceptible to CO, injury; and up to 15% CO, may improve the appearance of green vegetables and salad crops (Smith, 1938b; McGill, Nelson & Steinberg, 1966; Wang et al. 1971), though there may be undesirable effects on flavour (see below). Low oxygen, down to a level of 2.5% appeared not to influence loss of chlorophyll significantly in the experiments of Singh et al. (1972) on lettuce, although Stoll (1973b) recorded better retention in Savoy cabbage with 3% O,, even in the absence of CO,, and Kidd & West (1934) found very good retention, over a period of 74 months, in Bramley s Seedling apples, in pure N,; 0, and accumulated CO, being absent. Loss of carotene during the storage of carrots at 0-5 C is reduced by low oxygen (< 3%) (Baumann, 973). Softening occurs during the ripening of fruit in storage and continues to an unacceptable degree. In practice it is due in part to water loss, but we are concerned here with biochemical change, as, for instance, the enzymic breakdown of protopectin (Hulme & Rhodes, 1971). Softening is retarded in apples by CO, to an increasing extent as the concentration is raised to 12% and in some instances by reduced oxygen down to 2% (Tomkins, 1965; allowance must be made for the possible effects of reduced 0, accompanying the CO, increase in his experiments). The results of Kidd & West (1939) on Cox s Orange Pippin apples showed an appreciable effect of 2.5 yo O,, 5 yo CO, in retaining firmness, but they had previously found softening to be only slightly retarded by low oxygen alone (Kidd & West, 1935). Other changes in texture. Smith (19383) stated that the leaves of broccoli stored in 5 % O,, 15 yo CO, were brittle. Flaoour. The development of flavour in Cox s Orange Pippin is retarded by 2.5 yo O,, 5% CO,, a normal ripe flavour developing during subsequent marketing in air (Kidd & West, 1939; see also below). Off-Javours. A bitter flavour, caused by iso-coumarin, may develop during the storage of carrots. Its development is inhibited or reduced by reducing the ambient oxygen to 1*5-3% (Hansen & Rumpf, 1973) and stimulated by ethylene (Phan, 1973). On the other hand, low oxygen (3%) has a deleterious effect on the flavour of raspberries and strawberries stored in it. Increased CO,, referred to above as a means of retarding chlorophyll loss, may also have adverse effects. Smith (1938b) found a strong unpleasant odour in broccoli held in 5 % O,, 15 yo CO, and McGill et al. (1966) found spinach to be unacceptable after one week when stored in 137; CO,.

15 Biophysical principles of controlled atmosphere storage '63 Kidd & West (1939) commented on a slightly abnormal flavour in Cox's Orange Pippin apples stored in 5 % CO,, 16% 0,. Other relevant complaints of a physiological nature. One of the most serious complaints in the storage of apples is superficial scald, which Huelin & Murray (1966) suggested was caused by oxidation products of a-farnesene. The incidence of scald is considerably reduced by increasing the ambient carbon dioxide concentration over the range O-IO% or by reducing the oxygen concentration to 3 yo or below (Fidler & North, 1961). Brown or black discolorations in the flesh of various commodities can be associated wholly or in part with carbon dioxide injury. Brown heart in apples is an example; as is core-flush, a brown discoloration within the vascular region which develops in the senescent apple, probably following the decreased permeability of the integument, and increased concentration of carbon dioxide in the flesh, during normal storage in air (see Kidd & West, 1949a, b). Core breakdown in pears is a basically similar complaint. Both are associated with succinic acid accumulation (Hulme, 1956; Williams & Patterson, 1964) probably related to the inhibitory effect of increased CO, on succinic oxidase (Ranson, Walker & Clarke, 1957). Some varieties are more prone to the complaint than others (see above: ' Differences between atmospheres recommended as optimal for different varieties') and in these it is hastened and aggravated if the ambient concentration of carbon dioxide is increased above the level in air. On the other hand, the complaint can be lessened by reducing the ambient oxygen concentration, without permitting the accumulation of carbon dioxide, to a level which appreciably diminishes the output, and hence internal concentration of carbon dioxide. In practice this means reducing the ambient oxygen to 2-3 %. Pink or brown discolorations have been observed in mushrooms stored at low temperatures in enhanced CO, concentrations (Smith, 1964; 1965). Changes in composition. Oxygen concentration has a marked effect on carbohydrate metabolism. Nelson & Auchincloss (1933) found the sucrose synthesizing system in potato disks to be inoperative in the absence of oxygen, and concentrations of 3% and below delay the accumulation of sugars in potatoes stored at 0-1 "C (Samotus & Schwimmer, 1963; Workman & Twomey, 1970; Harkett, 1971). Hansen & Rumpf (1973) noted that the loss of sucrose from stored carrots was less in low oxygen concentrations, though the content of reducing sugars was lower than in air; a result in conformity with the effect of low oxygen on the sucroselreducing sugar balance recorded by Weichmann & Ammerseder (1973). Carbon dioxide has similarly marked effects-for example, 5% CO, prevents the accumulation of reducing sugars in potato tubers at 5 "C but increases the sucrose accumulation (Denny & Thornton, 1941). Barker (1936) found the initial rate of total sugar accumulation in potato tubers at I "C to be considerably reduced by 10% CO, though after 15 wk the sugar content in 10% CO, was higher than in air. As might be expected, acid metabolism is subject to influence by atmospheric composition. Accumulation of succinic acid has been mentioned above. Increased carbon dioxide leads also to an increase in citric acid in pears and a decrease in malic acid (Williams & Patterson,) In pome fruit, the climacteric is associated with the development of a malate decarboxylating system. This is almost prevented by 6 APB 7a

16 164 W. G. BURTON reducing the oxygen concentration to 3 %, and entirery prevented in lower concentrations (Hulme & Rhodes, 1971). Etliyleneproduction is oxygen dependent at low levels. No effect upon the rate of production by apples is observed until the ambient oxygen concentration is reduced below 8%. Ambient 2.5% 0, halves the rate (Burg, 1962). Continued development, break of dormancy and growth. Smith (1939) found the 'blowing' of broccoli to be prevented at 3.3 "C by 10% CO,, the effect being retained after 3 days subsequently in air at "C. On the other hand the sprout growth of potatoes at 10' is stimulated by increasing the concentration of ambient CO, to 2-4%, giving a concentration in thesap, optimal for growth, of about 2 x I O-~ M (Burton, 1958). The increased growth results in part from increased cell division but also from a marked increase in the length of the fully elongated cells. In view of this it is of interest that Barker & Morris (1937) found the elongation of cut asparagus at 5 "C to be inhibited by controlled atmosphere storage in which the CO, was varied from 5 to 10% and 0, from 5 to 15% and that Smith (1965) found elongation of the stipes of stored button mushrooms to be depressed by increased carbon dioxide. He also found increased carbon dioxide (8%) to delay the opening of the caps at 10 'C, but concluded that storage at o "C in air was more effective than controlled atmosphere storage at higher temperatures. Low concentrations of oxygen, optimally 2-4%, stimulate the start of growth in dormant potatoes at 10-20, and growth is optimal in about 5% 0, (Burton, 1968) which would give a concentration in the sap of about 5-6 x I O-~ M. Burton suggested that the stimulation of growth could result from the loss of a growth inhibitor, this being most rapid under anaerobic conditions; the observed optimum being compounded of this optimally anaerobic effect together with an oxygen requirement for growth - such as, for example, the oxygen requirement for carbohydrate mobilization. In non-dormant tubers the stimulation of growth by low oxygen becomes less noticeable and late in the storage season is replaced by an inhibitory effect (Burton, 1968). Rotting by pathogens such as the fungal rots of apples can be much reduced by controlled atmosphere storage, particularly at the higher levels of CO,. Fifty per cent CO, will, for instance, completely suppress fungal rotting of blackcurrants at 4.4 "C (see e.g. Smith, 1957). Most commodities will not tolerate such high levels of CO,, but useful control of apple rots is given by 8-1o%, as in the original unscrubbed controlled atmosphere stores. Low oxygen also exercises some control but this would be direct control of the pathogen only at very low levels - Shaw (1969) suggested that a concentration of I or even o.5yo might be necessary; see also Follstad (1966) - and damage to the commodity would be likely. In considering the effects of controlled atmosphere storage on rotting it must be remembered that a rot is the result of an interaction between pathogen and host. The atmosphere affects the pathogen, but it also affects the host, and the net effect may be beneficial or otherwise. Reduced fungal rotting of apples in controlled atmosphere storage probably results in the main from its effect in delaying ripening and resultant increased susceptibility of the fruit (Kidd et al. 1927), which means that 2-3 yo 0, can be beneficial despite there being no direct effect on the pathogen. Shaw (1969) concluded that the beneficial effects of s"/:, and

17 Biophysical principles of controlled atmosphere storage % CO, on Botrytis and Rhizopus rots of strawberries resulted from effects on the fruit, rendering them a substrate less favourable to the pathogens. CONCLUSIONS The general conclusions to be derived from the foregoing may be summarized as follows: Reduction of the oxygen concentration in the storage atmosphere as a means of reducing the rate of natural biochemical change in stored commodities is effective only at levels at which it has an appreciable effect on the terminal oxidases concerned. There is little evidence that the normal metabolism of vegetables and fruits is essentially linked to the functioning of low-oxygen-affinity oxidases, and it is only when we affect the operation of cytochrome-c oxidase that we begin to see marked effects on metabolism. Oxygen uptake by cytochrome-c oxidase is little affected until the concentration of oxygen at the active centres falls to a very low level. Estimates of the concentration at which a 50% reduction in functioning would be achieved vary from 7 x lo-* to 3 x M, in equilibrium, at usual storage temperatures, with about ~2~0 0, in the adjacent intercellular space. Control of the oxygen in the storage atmosphere must therefore achieve a level of less than 0.2% 0, in the bulk of the tissue if it is to be really effective, but at the same time a completely anaerobic state must be avoided. The ambient concentration necessary to produce these conditions varies with the permeability of the outer integument and tissues of different commodities, and even in one commodity variation between individuals is sufficient to render precise control of the internal atmosphere impossible; although to some extent the reduction in uptake associated with low oxygen levels buffers the internal variation in relation to the ambient concentration. In general, for most commodities for which a reduced level of oxygen in the storage atmosphere is beneficial- as described in the foregoing section - ambient concentrations within the range 1-3 yo 0, are necessary. Apart from effects on the normal metabolism, a reduced concentration of oxygen influences reactions resulting in discolorations at cut surfaces. These are catalysed by low oxygen-affinity enzymes and hence can be controlled if the ambient oxygen concentration is reduced below about 5 yo. Increasing the carbon dioxide in the storage atmosphere has an additive effect upon internal concentration, modified by any change in carbon dioxide output which is caused. In fact ambient 10% CO,, typically leading to a two- to four-fold increase in internal concentration, and about 6-8 x I O-~ M CO, in the sap, at usual storage temperatures, can about halve the rate of output, with consequent re-establishment of biochemical equilibria resulting also in an approximate halving of oxygen uptake. Ambient 10'7, CO, thus has effects upon some aspects of metabolism comparable to those caused by reducing the ambient oxygen concentration to, say, 2%, and can have similarly beneficial effects in retarding undesirable changes, as described in the foregoing section. A high level of internal carbon dioxide, however, whether occurring naturally in the course of storage in air, or induced by an increased ambient concentration, can also have harmful effects, resulting in such symptoms as brown-heart and 5-2

18 166 W. G. BURTON core-flush in apples and core-breakdown in pears. Some varieties are so susceptible to such damage that an increase in CO, sufficient to give worthwhile reductions in undesirable changes, has associated hazards which outweigh the advantages. It is arguable that such varietal differences may result from differences in anatomy and tissue permeability, rather than from biochemical differences at a single cell level. REFERENCES BARKER, J. (1936). The influence of the carbon dioxide and oxygen in the atmosphere on the sugar content and sprouting of potatoes. Report of the Food Investigation Board for 1935, pp BARKER, J. & MORRIS, T. N. (1937). The storage of asparagus. Report of the Food Investigation Board for 1936, pp BAUMANN, H. (1973). Preservation of carrot quality in various storage conditions (Summary). International Society for Horticultural Science. Summaries of symposium on vegetable storage, Freising-Weihenstephan, Sept. 37, BRXNDLE, R. (1968). Die Verteilung der Sauerstoffkonzentrationen in fleischigen Speicherorganen (Apfel, Bananen und Kartoffelltnollen). Bericht der Schweizerischen botanischen Gesellschaft 78, BURG, S. P. (1962). The physiology of ethylene formation. Annual Review of Plant Physiology 13, BURG, S. P. & BURG, E. A. (1965). Gas exchange in fruits. Physiologia Plantarum 18, BURTON, W. G. (1950). Studies on the dormancy and sprouting of potatoes. I. The oxygen content of the potato tuber. New Phytologist 49, BURTON, W. G. (1958). The effect of the concentrations of carbon dioxide and oxygen in the storage atmosphere upon the sprouting of potatoes at 10 "C. European Potato Journal I, 45-57, BURTON, W. G. (1965). The permeability to oxygen of the periderm of the potato tuber. Journal of Experimental Botany 16, BURTON, W. G. (1968). The effect of oxygen concentration upon sprout growth on the potato tuber. European Potato Journal 11, BURTON, W. G. (1974). The oxygen uptake, in air and in 5 yo 02, and the carbon dioxide output, of stored potato tubers. Potato Research 17, BURTON, W. G. & SPRAGG, W. T. (1950). A note on the intercellular space of the potato tuber. Neeu Phytologist 49, CRAFT, C. C. (1963). Respiration of potatoes as influenced by previous storage temperature. American Potato Journal 40, DENNY, F. E. & THORNTON, N. C. (1941). Carbon dioxide prevents the rapid increase in the reducing sugar content of potato tubers stored at low temperatures. Contributions. Boyce Thompson Institute for Plant Research 12, DOSTAL, H. C. & LEOPOLD, A. C. (1967). Gibberellin delays ripening of tomatoes. Science 158, DRAWERT, F., HEIMANN, W., EMBERGER, R. & TRESSL, R. (1968). Uber die Biogenese von Aromastoffen bei Pflanzen und Friichten-111. Gaschromatographische Bestandaufnahme von Apfel-aromastoffen. Phytochemistry 7, EAMES, A. J. & MACDANIELS, L. H. (1947). An Introduction to Plant Anatomy. New York: McGraw-Hill. FIDLER, J. C. & MA", G. (1972). Refrigerated Storage of Apples and Pears - a Practical Guide. Commonwealth Agricultural Bureaux. FIDLER, J. C. & NORTH, C. J. (1961). Effect of various pre-treatments, and of concentration of oxygen in storage, on scald. Bulletin of the International Institute of Refrigeration, Annexe , pp FJDLER, J. C. & NORTH, C. J. (1967). The effect of conditions of storage on the respiration of apples. I. The effects of temperature and concentrations of carbon dioxide and oxygen on the production of carbon dioxide and uptake of oxygen. Journal of Horticultural Science 4,

19 Biophysical principles of controlled atmosphere storage 67 FOLLSTAD, M. N. (1966). Mycelial growth rate and sporulation of Alternaria tenuis, Botrytis cinerea, Cladosporum herbarum and Rhizopus stolonifer in low oxygen atmospheres. Phytopathology 56, FRENKEL, C., KLEIN, I. & DILLEY, D. R. (1968). Protein synthesis in relation to ripening of pome fruits. Plant Physiology, Lancaster 43, I I 53. GERARD, R. W. (1931). Oxygen diffusion into cells. Biological Bulletin. Marine Biological Laboratory, Woods Hole, Mass. 60, 245. GODDARD, D. R. (1947). The respiration of cells and tissues. Section 6 (pp ) of HBber, R. (1947). Physical Chemistry of Cells and Tissues, London: Churchill. GORTER, A. & NADORT, W. (1941). Composition of gas in the intercellular spaces of potatoes. Proceedings of the Academy of Science, Amsterdam 4, I I 12. IIANSEN, H. & RUMPF, G. (1973). Storage of carrots: the influence of the storage atmosphere on taste, wastage and contents of sucrose, fructose and glucose (Summary). International Society for Horticultural Science. Summaries of symposium on vegetable storage, Freising- Weihenstephan, Sept. 37, HARDY, J. K. (1949). Diffusion of gases in fruit: the solubility of carbon dioxide and other constants for Cox s Orange Pippin apples. Report of the Food Investigation Board for 1939, pp HARKETT, P. J. (1971). The effect of oxygen concentration on the sugar content of potato tubers stored at low temperature. Potato Research 14, HUELIN, F. E. & MURRAY, K. E. (1966). a-farnesene in the natural coating of apples. Nature, London 210, HULME, A. C. (1951). Apparatus for the measurement of gaseous conditions inside an apple fruit. Journal of Experimental Botany 2, HULME, A. C. (1956). Carbon dioxide injury and the presence of succinic acid in apples. Nature, London 178, HULME, A. C. & RHODES, M. J. C. (1971). Pome Fruits. Chapter 10 (pp ) of Vol. 2 of The Biochemistry of Fruits and their Products, edited by A. C. Hulme. London: Academic Press. KAESS, G. (1943). Gartenbaueuissenschaft 17, 591, quoted from Stoll (1973~). KIDD, F. & WEST, C. (1927~). A relation between the respiratory activity and the keeping quality of apples. Report of the Food Investigation Board for 1925, 1926, pp KIDD, F. &WEST, C. (1927b). A relation between the concentration of oxygen and carbon dioxide in the atmosphere, rate of respiration, and length of storage life of apples. Report of the Food Investigation Board for 1925, 1926, pp KIDD, F. & WEST, C. (1934). Injurious effects of pure oxygen upon apples and pears at low temperatures. Report of the Food Investigation Board for 1933, pp KIDD, F. & WEST, C. (1935). Gas storage of apples. Report of the Food Investigation Board for 1934, PP KIDD, F. & WEST, C. (1939). The gas-storage of Cox s Orange Pippin apples on a commercial scale. Report of the Food Investigation Board for 1938, pp KIDD, F. & WEST, C. (1949~). Resistance of the skin of the apple fruit to gaseous exchange. Report of the Food Investigation Board for 1939, pp KIDD, F. & WEST, C. (I949b). Carbon dioxide injury in relation to the maturity of apples and pears. Report of the Food Investigation Board for 1939, pp KIDD, F., WEST, C. & KIDD, M. N. (1927). Gas storage of fruit. Special Report of the Food Investigation Board, Department of Scientific and Industrial Research 30. MCGILL, J. N., NELSON, A. I. & STEINBERG, M. P. (1966). Effects of modified storage atmosphere on ascorbic acid and other quality characteristics of spinach. Journal of Food Science 31, MAPSON, L. W. & BURTON, W. G. (1962). The terminal oxidases of the potato tuber. Biochemical Journal 82, MEIGH, D. F., JONES, J. D. & HULME, A. C. (1967). The respiration climacteric in the apple. Production of ethylene and fatty acids in fruit attached to and detached from the tree. Phytochemistry 6, I I 5. NELSON, J. M. & AUCHINCLOSS, R. (1933). The effects of glucose and fructose on the sucrose content of potato slices. Journal of the American Chemical Society 55,

20 I 68 W. G. BURTON PHAN, C. T. (1973). Biochemical studies on the development of bitterness in stored carrots (Summary). International Society for Horticultural Science. Summaries of symposium on vegetable storage, Freising-Weihenstephan, Sept. 3-7, KANSON, S. L., WALKER, D. A. & CLARKE, I. D. (1957). The inhibition of succinic oxidase by high CO, concentrations. Biochemical Journal 66, 57~. RHODES, M. J. C. (1970). The climacteric and ripening of fruits. Chapter 17 (pp ) of Vol. I of The Biochemistry of Fruits and their Products, edited by A. C. Hulme. London: Academic Press. SAMOTUS, B. & SCHWIMMER, S. (1963). Changes in carbohydrate and phosphoric compounds of potato tubers during storage in nitrogen. Journal of Food Science 28, SHAW, G. W. (1969). The effects of controlled atmosphere storage on the quality and shelf life of fresh strawberries with special reference to Botrytis cinerea and Rhizopus nigricans. Ph.D. Thesis, University of Maryland. SINGH, B., YANG, C. C. & SALUNKHE, D. K. (1972). Controlled atmosphere storage of lettuce. I. Effects on quality and the respiration rate of lettuce heads. Journal of Food Science 37, Effects on biochemical composition of the leaves.journa1 of Food Science 37, SMITH, W. H. (1938~). Anatomy of the apple fruit. Report of the Food Investigation Board for 1937, PP SMITH, W. H. (19386). The storage of broccoli. Report of the Food Investigation Board for 1937, PP SMITH, W. H. (1939). The gas storage of broccoli. Report of the Food Inwestigation Board for 1938, pp SMITH, W. H. (1947). A new method for the determination of the composition of the internal atmosphere of fleshy plant organs. Annals of Botany (N.S.) 11, SMITH, W. H. (1957). Accumulation of ethyl alcohol and acetaldehyde in blackcurrants kept in high concentrations of carbon dioxide. Nature, London 179, SMITH, W. H. (1964). The storage of mushrooms. Report of the Ditton Laboratoryfor ,18. SMITH, W. H. (1965). Storage of mushrooms. Report of the Ditton Laboratory for , 25. SPECTOR, W. S. (1956) (editor). Handbook of Biological Data: Technical Report of the Wright Air Development Center; STOLL, K. (1972). Lagerung von Fruchten und Gemiisen in kontrollierter Atmosphare. Mitteilungen der Eidgeniissichen Forschungsanstalt fiir Obst-, Wein- und Gartenbau, Wadenswil, Flugschrift 77. STOLL, K. (1973~). Tabellen zur Lagerung von Friichten und Gemusen in kontrollierter Atmosphare. Mitteilungen der Eidgenossichen Forschungsanstalt fiir Obst-, Wein- und Gartenbau, Wadenswil, Flugschrift 78. STOLL, K. (1973 b). Storage of vegetables in modified atmospheres (CA). International Society for Horticultural Science. Summaries of symposium on vegetable storage, Freising-Weihenstephan, Sept. 37, STREBEYKO, P. (1965). The theory of porometer. Physiologia Plantarum 18, TOMKINS, R. G. (1965). Small-scale storage trials. Report of the Ditton and Covent Garden Laboratories for , pp TOMKINS, R. G. & MEIGH, D. F. (1968). The concentration of ethylene found in controlled atmosphere stores. Report of the Ditton Laboratory for , pp WANG, S. S., HAARD, N. F. & DIMARCO, G. R. (1971). Chlorophyll degradation during controlled atmosphere storage of asparagus. Journal of Food Science 36, WARDLAW, C. W. & LEONARD, E. R. (1936). Studies in tropical fruits. I. Preliminary observations in some aspects of development] ripening and senescence, with special reference to respiration. Annals of Botany, London 50, WARDLAW, C. W. & LEONARD, E. R. (1939). Studies in tropical fruits. IV. Methods in the investigation of respiration with special reference to the banana. Annals of Botany, London (N.S.) 3, 27. WEICHMANN, J. & AMMERSEDER, E. (1973). Influence of CA-storage condition on carbohydrate changes in carrots (Summary). International Society for Horticultural Science. Summarics of symposium on vegetable storage, Freising-Weihenstephan, Sept. 3-7, WILLIAMS, M. W. & PATTERSON, M. E. (1964). Non-volatile organic acids and core breakdown of Bartlett pears. Journal of Agricultural and Food Chemistry 12, WORKMAN, M. & TWOMEY, J. (1970). The influence of storage on the physiology and productivity of Kennebec seed potatoes. American Potato Journal 47,