THE EFFECT OF OXYGEN AND OZONE
ON
MICROBIAL RESPIRATION
A thesis submitted to The University of New South Wales
for the degree of Doctor of Philosophy
by
GORDJN ALEXANDER BEECH
School of Biological Technology,
September, 1969, (il
ACKNOWLEDGEMENTS
I wish to express :my sincere gratitude to :my supervisors,
Associate Professor F,J. Moss and Associate Professor G.H. Roper for their continual support and encouragement through.out this work. The award of a Research Fellowship and other financial support from the
New South Wales Air Pollution Committee is gratefully acknowledged.
The work was carried out in the laboratory of the School of Biological
Technology, The University of New South Wales and thanks are due to
Professor B.J. Ralph, Head of the School, for much helpful advice and support. Many stimulating and fruitful discussions were held with
Dr. P.A.D. Rickard, Dr. P.L. Rogers and Mr. P.P. Gray. Invaluable assistance in the maintenance arid operation of the continuous culture apparatus was provided by Mr. F.E. Bush and Mr. H. Lo~h. The staff of the Biological Sciences Workshop, under Mr, W. Wynne, deserve special thanks for their expert assistance in the construction and maintenance of the apparatus, and for construction of the ozone generator. Mrs, B.
Lorbergs prepared the excellent photographic prints, (ii)
SUMMARY
The response of the yeast Candida utilis to growth at different controlled oxygen tensions was studied. C. utilis was grown in chemostat culture :i.n which the oxygen tension, pH, temperature and dilution rate of the culture were controlled. A series of 12 steady states was studied over which the controlled dissolved oxygen value was varied from 400 µM to a very low value b~low the limit of' measurement with the Mack.ereth oxygen electrode. At each steady state measurements were made of the dry weight of the culture, cytochrome concentration in the cells, rates of uptake by the culture of oxygen and glucose and rates of production of ethanol, carbon dioxide and acid. A similar study was made of a series of three steady states in which oxygen tension was not controlled but oxygen solution rate was set at low but progressively increasing values. At measurable oxygen tensions there was no dependency of oxygen uptake on dissolved oxygen, except at very high oxygen tensions, where abnor.mally high oxygen uptake rates were recorded. At immeasurably low oxygen tensions, the rate of oxygen uptake varied directly with o:xygen solution rate. The culture exhibited exclusively oxidative metabolism at all controlled dissolved oxygen values above 1, 5 µM. Below this, fermentation of glucose to ethanol and carbon dioxide occurred. The rate of production of ethanol was greatest at the lowest oxygen solution rate studied, in which fermentation was the major component of energy metabolism. The concentration of cytochromes in the cells was independent of o.:xygen tension except at very low values (0,15 µMand (iiil
below), 'Where maximal cytochrome levels were recorded. The cytochrome concentration was low at low oxygen solution rates where fermentation predominated.
In two experiments, the behaviour of C, utilis was studied in a chemostat culture during the transient period following a step change from a high to a low oxygen tension. A smooth transition to a new steady state did not follow, but fluctuations occurred in the metabolic parameters of the culture. 'Ihe time course of the changes during the transient period and the length of time which elapses before a new steady state is reached are related to the degree of oxygen limitation which the step change imposed.
The results of these experiments are discussed in the light of current knowledge of the role of o:xygen in metabolic regulation. It is considered unlikely that o:xygen operates at the molecular level as a regulator of cytochrome biosynthesis apart from the requirement of o:xygen for its initiation.
The effect of ozone on C. utilis was studied in resting cell suspensions and continuous culture. Ozone was found to have lethal effects on cells of C, 11tilis, the internal structure being disrupted and the cytochrames destroyed. Growth conferred on C. utilis a much greater resistance to the toxic effects of ozone and ozone was found to stimulate the rate of oxygen uptake. 'Ihe high reactivity of ozone does not permit its selective effect as a mutagen. (iv}
GLOSSARY OF SYMBOLS AND TERMS
Cytochrome A: Concentration of cytochrome a/a3 , having an a absorption maximum at 600 mµ, expressed as percent of the arbitrary standard.
Cytochrome B: Concentration of b-type cytochromes, having an a absorption maximum at 560 mµ, expressed as percent of the arbitrary standard.
Cytochrome C: Concen;:;ration of £_-type cytochromes, having an a absorption maximum at 548-550 mµ expressed as percent of the arbitrary standard.
Rate of uptake of oxygen in millimoles/g.dry wt/hr.
Rate of production of carbon dioxide in millimoles/g.dry wt/hr
Rate of uptake of glucose in millimoles/g.dry wt/hr.
Rate of production of ethanol in millimoles/g.dry wt/hr,
X Concentration of cell mass in g ,dry weight/litre of culture. l dx . -l µ Specific growth rate, i" dt, in hr ,
C Fraction of carbon in dried cell mass.
Fraction of glucose carbon used which is fixed into cell substance. dx y The yield constant, equal to ds • s Concentration of substrate in the continuous culture vessel,
Concentration of substrate in the medium feed, (V}
CONTENTS
ACKNOWLEDGEMENTS (i}
SUMMARY (ii}
GLOSSARY OF SYMBOLS AND TERMS (iy}
CONTENTS (v}
1. l:NTRQDUC'l'ION
1.1 REGULATION OF METABOLISM IN MICROORGANISMS
1.1,1 Adaptation in Microorganisms 1
1,1.2 Regulation of Enzyme Activity 2
1.1,3 Regulation of Enzyme Synthesis 4
l. 2 'FUNCTIONS OF OXYGEN IN METABOLIC REGULATION IN
MICROORGANISMS
1,2,l fuygen and the Tie-velopment of Yeast Mitochonfu>ia 5
l,2,2 Role of fuygen in Relation to Cytochromes 6
1.2.3 Role of Oxygen in Relation to Tricarboxylic Acid
(TCA} Cycle Enzymes 9
1.2. 4 Ef-fects of Excess Oxygen on Microorganisms 10
l.3 OZONE
l,3.l Occurrence ana Reactions with Organic Material ll
1,3.2 Effects on Plants ana Animals 12
l,3,3 Effects on MicroOl'g~iSllls 14 (-vil
1.4 CONTINUOUS CULTURE OF MICROORGANISMS
1, 4.1 Theoretical Considerations 15
1\4.2 Value of Continuous Culture as a ReseaTch Tool 18
2. APP AM.TUS AND EXPERIMENTAL METIIDDS
2.1 THE ORGANISM 20
2,2 ROUTINE MICROBIOLOGICAL PROCEDURES
2.2,1 Maintenance of Cultures 20
2,2.2 Preparation of Inocula for Continuous Culture
Experiments 20
2.3 CULTURE MEDIUM
2,3,1 Composition of the Culture Mea.i1l!ll 21
2,3,2 Preparation of the Culture Medium 22
2,3.3 Sterilisation of the Culture Mea.ium 23
2.4 CONTINUOUS CULTURE APPARATUS
2, 4,1 The Culture Ves·sel 25
2. 4.2 Supply- of Medium to the Culture 'Vessel 26
2,4.3 Collection of Cells and Products 26
2, 4. 4 Gas Flow 27
2,4;5 Measurement of Oxygen Tension 28
2.4.6 Control of Oxygen Tension 29
2. 4. 7 Control of Temperature 30
2,4.8 ContTol of pff 30 (-vii}
2,_ 4,2 ContTol of Cell Density 31
2, 4,10 Ste-rilisation of the Continuous Culture Apparatus 32
2. 4 ,TI Ope-ration of the Continuous Culture Apparatus
2,4,TI.l The Setting~-up of a Continuous Culture 33
2~ 4,TI,2 Sampling PToceaures 35
2.5" ANALYSIS OF THE CULTURE
2,5,l Measurement of CytochTa:ine Concentration
2,5.l,l Reflectance Spectroscopy 37
2.5,l,2 Calculation of Cytochrome Concentration 38
2.5.2 Measurement of Cell Dry Weight of the Culture 38
2.5,3 Measurement of Glucose Concentration 39
2,5,4 Measurement of Ethanol Concentration 40
2,5,5 Measurement of the Viability of the Culture 41
2,5,6 Gas .Analysis
2,5,6,l Measurement of Oxygen ana Carbon Dioxiae 43
2,5,6,2 Measurement of Ozone 43
2, 5 ,1 Ex~ssion of Metabolic Parameters of the Culture 44
2,6 GENERATION OF OZONE
2,1 ELECTRON MI'CROSCOPY
3, EKPERIMENTAL'---'RESULTS
3,l GROWTH IN STEADY STATE CONTINUOUS CULTURE AT VARIOUS
CONTROLLED LEVELS OF DISSOLVED OXYGEN (yiiil
3.2 GROWTff IN STEADY STATE CONTINUOUS CULTURE AT VARIOUS
LEVELS OF OXYGEN SOLUTrON RATE 53
3.3 THE TRANSrENT STATE FOLLOWING A STEP CID\NGE FROM HIGH
TO LOW DISSOLVED OXYGEN
3,3.J. The Natuxe of the Imposed Disturbance 54
3.3.2 Trans·ient state 1'ollowing a Step Change in the
Controlled Level of Dissolved Oxygen
3.3.3 Transient State following a Step Change from a
Controlled Dissolved Oxygen Value of 235 µM to
a Fixed Low Oxygen Solution Rate 64
3. 4 THE EFFECTS OF OZONE ON C, UTrLrs
3. 4.J. Effects of Ozone on the Cytocb.Tomes of a Resting
Cell Suspension 68
3.4,2 Effects of Ozone on Cytocb.Tomes and Viability of
a Non-Growing Culture 70
3. 4.3 Effects of Ozone on Cytocb.Tames and Growth Rate
of a Turbidostat Culture 72
3. 4, 4 Effects of Ozone on Cell Dens-rty, Viability,
Cytocb.Tomes; Metabolism and Cell Fine Structure
of a Chem.ostat Cultuxe 75 4, DISCUSS!:ON,~CONCLUSIONS
4,1. COMPARISON OF STEADY STATE RESPONSE OF C, UTII:iIS TO
DISSOLVED OXYGEN VALUE. AND OXYGEN SOLUTION RATE 8l
4. 2 BEHAVIOUR OF C, UTI-I,IS DURING ADAPTATION TO A LOW OXYGEN ENVIRONMENT 86
4,3 DAMAGE TO C, 5. REFERENCES 91 .APPENDIX I EQUATIONS USED IN CALCULATION OF "Q" VALUES (xl APPENDIX II SAMPLE CALCULATION OF PREDICTED CYTOCHROME A AND DRY WEIG BT YALUES IN TABLE 3 ,. 6 (xiii} PUBLICATION SUBMITTED IN SUPPORT OF THESIS (xivl .l. INTRODUCTION l.l REGULATION OF METABOLISM IN MICROORGANISMS l.1.1 Adaptation in Microorganisms One of the most striking properties of microorganisms is the frequent ability of individual species to grow over a wide range of differing environmental conditions. As in all living forms, adaptation in microorga.nisms has two major facets. One is mutation, or random change in the genetic material of the cell which leads to a permanent inherited change in some property of the cell. If this change is advantageous to the cell's survival in a particular environment, adaptation has occurred. The other facet is a physiological response of the organism to some environmental change without the genetic material of the cell being altered. Because of their very small size and relatively simple level of organisation in relation to the rest of the living world, the physiological changes which bacterial cells or populations of cells undergo in response to a change in the environment may be identified at the biochemical level. Thus adaptation in microorga.nisms is equated with regulation of metabolism. The various aspects of adaptation in microorga.nisms was the subject of the 3rd Symposium of the Society for General Microbiology (1953). Current ideas on metabolic regulation are centred around the regulation of enzyme activity and the regulation of enzyme synthesis. 2, 1.l.2 Re5ulation of Enzyme Activity At the simplest level, the activity of an enzyme is determined by the availability of its substrate and also of any essential cofactor, However of greater importance to metabolic control is the activation and inhibition of an enzyme by metabolites different from those involved in the reaction catalysed by the enzyme. Early observations of specific regulation of enzyme activity by metabolic products were made on biosynthetic enzymes. An example is amino acid biosynthesis, which is commonly regulated in bacteria by feedback inhibition of the first enzyme of the pathway by the end-product. This phenomenon was reported in the pathway of histidine synthesis by Ames, Martin and Garry (1961). Thus, although only the first enzyme is directly inhibited, the activity of the entire pathway is reduced. Feedback inhibition of amino acid pathways has been reviewed by Cohen (_1965}. End product inhibition of biosynthetic pathways constitutes a system which circumvents unnecessary expenditure of energy on biosynthetic processes when an adequate supply of the end product is available. The feedback inhibitor is often chemically dissimilar to the substrate of the inhibited enzyme. Inhibition of this type was described by Monod, Changeux and Jacob (19631 as allosteric inhibition. It was postulated that there is combination of the inhibitor molecule with the enzyme at a site other than the active site, resulting in altered conformation of the enzyme protein, and a change in the affinity of the enzyme for its substrate (Monod, Wyman and Changeux, 1965). Gerhardt and Schachman (1965) have shown that aspa.rtate 3, tra.nsca.rba.Iey'lase consists of two types of subunits, one of which (the substrate - binding subunit) carries the active site of the enzyme while the other (the regulatory subunit l carries the binding site for the feedback inhibitor, in this case CTP. Evidence exists (Gerhardt and Schachman, 1968) for the existence of two different conformational states of this enzyme in the presence or absence of the speci fie ligands • End-product inhibition operates on branched biosynthetic pathways as well as those leading to the formation of only one end-product. In the case of a bifurcating pathway the enzyme at the branch point often occurs as two distinct isoenzymes, each of which is sensitive to inhibition by a different end..-product. This system enables one branch of the pathway to be inhibited while the other continues to operate. In addition to this mechanism, there is of'ten inhibition of the first enzyme a:f'ter the branch point, making the feedback inhibition of branched pathways a complex pattern of allosteric regulation, In addition to enzymes of biosynthetic pathways, the activities of several key enzymes of the central metabolic pathways a.re subject to allosteric regulation. Phosphofructokinase (Blangy, Bue and Monod, 1968} is inhibited by phosphoenolpyruvate and also by ATP or citrate, and is activated by ADP, AMP or fructose 1,6-diphosphate. Isocitrate lyase in Escherichia coll is also inhibited by phosphoenolpyruvate (Ashworth and Konibay, 19631 and ATP. ADP and AMP are activators of this enzyme. Thus the ATP/ADP ratio plays an important role in regulation of metabolic activity. 4. l.l.3 Regulation of Enzyme Synthesis While regulation of enzyme activity enables fine control of metabolic balance, a more coarse control at the level of enzyme synthesis ensures that the cell contains only those enzymes required for growth in a particular chemical environment, Thus amino acids and energy are not wasted on the synthesis of enzymes which will be non-functional. The synthesis of certain enzymes is known to be induced by their substrates. Repression of enzyme synthesis also occurs and this may be caused by the end-product of a biosynthetic pathwizy- in which the enzyme functions or, in the case of a catabolic enzyme, by a metabolic intermediate which is a catabolite of the pathwizy- in which the enzyme operates. Studies on the regulation of enzyme synthesis and the mechanism by which it occurs have largely involved the synthesis of S-galactosidase in E. coli. Studies on this system led to the development of the operon model of Jacob and Monad (1961) which is now widely accepted, This model explains induction and repression on the basis of action of inducers and repressors at the genetic level in controlling expression of genes responsible for initiation of synthesis of specific enzymes or groups of enzymes. The operon theory and its subsequent experimental confirmation have been sUI1DI1arised and reviewed by Clarke and Lilly (1969). During growth, the overall metabolic pattern in a microbial cell depends on the interaction of the various factors controlling enzyme activity and those controlling enzyme synthesis. The regulation of synthesis of ' 5. respiratory structures in microorganisms has been the object of much study and present views relate such regulation to the concentrations of oxygen and glucose present during growth. The "glucose effect" of repression of synthesis of numerous enzymes has been discussed by Moses and Prevost (1966). A considerable body of literature has accumulated relating to the effect of glucose on synthesis of yeast respiratory enzymes and mitochondria. This field has been reviewed by Moss, Rickard, Beech and Bush (1969 ) . In aerobic organisms, the part pleyed by oxygen in metabolic regulation has been a point of considerable interest for some time. The general subject of oxygen as a regulator of microbial growth and metabolism has been extensively reviewed by Wimpenny (1969), 1.2 FUNGrIONS OF OXYGEN IN MEI'ABOLIC REGULATION IN MICROORGANISMS l.2 .1 O.xygen and the Development of Yeast Mitochondria Several studies of yeast with the electron microscope have shown that mitochondrial structure is absent in anaerobically grown yeast, but appears when the culture is aerated (Linnane, Vi tols and Nowland, 1962; Polakis, Bartley and Meek, 1964; Wallace and Linnane, 1964; Linnane, 1965). It was reported by Tuns tan off and Bartley (1964) that mitochondria and respiratory activity occurred in anaerobic cultures of Saccharonwces cerevisiae using galactose as carbon source. However Somlo and Fuk.uhara (1965) in repeating these experiments found that when precautions are taken to eliminate all traces of oxygen, the levels of respiratory enzymes 6. present in the cells were very small. They showed that only traces of oxygen are required for the development of cytochrome spectra similar to those of fully aerated cells. Recent work by Plattner and Schatz (1969), however, indicates the presence of non-respiring, mitochondria - like structures in anaerobically grown cells of s. cerevisiae. These structures, which have been termed pro-mitochondria by the authors, were observed with the electron microscope in frozen-etched specimens. The authors do not indicate, however, whether these promitochondria were visible in positively stained thin sections of the cells. Roodyn (1966) has demonstrated an in vitro requirement of oxygen by a system synthesising yeast mitochondrial protein. Thus mitochondrial development mey be oxygen dependent although only trace amounts are needed for its initiation. l, 2 ,2 Role of 0.:xygen in Relation to Cytochromes When adapted to aerobic conditio~ yeasts display a classical cytochrome system comparable to the mammalian system and consisting of cytochromes §., ~ 3, °Q_, £_ and c1 (Slonimski, 1953; Mackler, Collipp, Duncan, Rao and Huenekers, 1962) . Cytochrome ~, catalase and cytochrome £_ peroxi dase are other haem-containing compounds present in aerobic yeast (Sherman, 1965). The presence of cytochrome J?.i has been reported in anaerobic yeast by Chaix and Labbe (_1.965}. There are many reports of observations of maximal levels of cytochrome in organisms grown under conditions of low oxygen. Oxygen repression of tetrapyrrole and cytochrome synthesis has been reviewed by Lascelles (1961, 7. c.oncenl-'VD-t-t OV\, 196 41 and Smith (1961 l , Repression of cytochrome £._ by oxygen has been " reported in Pseudomonas fluorescens (Lenhoff, Nicholas and Kaplan, 1956}, Microcoecus denitrif'ica.ns (Chang and La.scelles, 1963; Verhoeven and Takeda, 1956), and E, coli (Wimpenny, Ranlett and Gray, 19631, Studies by Moss using continuous culture have shown that in E, coll (Moss, 1952) and Aerobacter aerogenes (Moss, 19561, cytochrome ~ occurred in maximum concentration in the presence of limited oxygen. Studies of Ps. fluorescens (Rosenberger and Kogut, 1958), Haemophilus influenzae (White, 1962) and Bacillus megatherium (Herbert, 1965 l have also shown an increase in cytochrome ~2 when these organisms are grown at low oxygen, A requirement of oxygen for synthesis of respiratory pigments has been shown in StaphYlococcus epidermidis which, although facultatively aerobic, cannot synthesise haem anaerobically. (Jacobs and Conti, 1965 ; Jacobs, McClosky and Jacobs, 1967), There appears to be a requirement for oxygen fo:r,h.aem biosynthesis in this organism as is the case for haem biosynthesis in higher organisms (Goldfine and Bloch, 1963; Sano and Granick, 1961). Anaerobically, S, epidermidis accumulates coproporphyrinogen but has no cytochrome ~ and only traces of cytochromes ~ and £, all of which are present in aerobic cells, Addition of haem to anaerobically grown cells was found to result in formation of cytochromes b;:i_ and £ but not a, and changing to aerobic growth led to increases in cytochromes b:L and o and finally the formation of cytochrome ~ (Frerman and White, 1967). Investigations into the site of action of oxygen in haemoprotein synthesis have also been made by Yeas and Drabkin (1957), These authors 8. showed by use of c14 labelled glycine that neither the protein nor the haem group of cytochrome £.. are formed in anaerobically grown yeast, but are induced by oxygen. They also used p-fluorophenylalanine to specifically inhibit protein synthesis during adaptation of anaerobic yeast to oxygen and found that free haem did not accumulate, indicating that synthesis of the haem group was associated with synthesis of the protein moiety. Some facultative bacteria do appear to be able to form cytochrome pigments anaerobically. E. coll grown under anerobic conditions (Gray, Wimpenny, Hughes and Mossman, 19661 was shown to possess many properties characteristic of aerobic cells, including the cytochromes. However Somlo and Fukuhara (1965) have refuted similar findings with yeast by Tustanoff and Bartley (1964} on the basis of failure to exclude the last traces of oxygen from an~robic cultures and in the light of this, the above report . A cannot be accepted with.out some reservation. While much evidence points to the requirement of oxygen for cytochrome synthesis, oxygen in mild excess has been shown to retard steps in porphyrin biosynthesis. a-amino levulinic acid synthetase is repressed by oxygen in Rhodospirillum spheroides (Lascelles, 1960) and Spirillum itersonii (Clark- Walker, Rittenberg and Lascelles, 1967). Also proto porphyrin synthesis in avian red cells is suppressed by oxygen pressures higher than 7% of the atmospheric value (Falk, Porra, Brown, Moss and Larminie, 1959; Porra and Falk, 1964}, the oxygen - sensitive step being conversion of porphobilinogen to uroporphyrinogen (Falk and Porra, 1964}. 2_, l,2,3 Role of O:xygen in Relation to Trica.rbo:xylic Acid (TCAl Cycle Enzymes The primari]y oxidative fm1ction of the TCA cycle suggests that the activities and levels of its component enzymes might be affected by o:xygen, In adaptation of yeast to aerobic conditions, activities of aconitase, fumarase and isocitrate dehydrogenase have been shown to increase (Hirsch, 1952; Slonimski and Hirsch, 19521. O:xygen is also fom1d to induce TCA cycle enzymes in Bacillus cereus (Schaeffer, 1952} and Pasteurella pestis (Englesberg and Levy, J.955). The latter workers also showed an increase in pyruvate oxidase, phosphotransacetylase and acetokinase in aerobic cultures as well as a substantial decrease in the levels of the g]yco]ytic enzymes 3-phosphog]yceraldehyde dehydrogenase and phosphofructokinase. Studies of other nutritional conditions on the levels of TCA cycle enzymes in E. c oli (Gray, Wimpenny, Hughes and Mossman, 1966; Gray, Wimpenny and Mossman, 1966) led to the suggestion that both catabolite repression and oxygen control the biosynthesis of these enzymes. Wimpenny and Necklen (unpublished results, cited by Wimpenny, J.9691 have found that in E, coli and A. aerogenes grown in continuous culture, synthesis of TCA cycle enzymes is maximal at high aeration rates and at a more positive Eh value than that which produces optimum cytochrome formation. This result has led them to the conclusion that synthesis of cytochromes and synthesis of TCA cycle enzymes are apparent]y controlled separate]y. _]_Q. 1.2.4 Effects of Excess Oxygen on Microorganisms Toxicity of oxygen at high concentrations is a phenomenon which has been observed in all forms of life, and the subject has been reviewed comprehensively by Haugaard (1968}. Moore and Williams (19lll first observed inhibition of gt'OWth of various bacteria at high oxygen tensions. More recent reports of the effects of hyperbaric oxygen on bacteria include those of Wiseman, Violago, Roberts and Penn (19661 and ZoBell and Hittle (1967}. Several authors have offered suggestions on the mechanism of oxygen toxicity, which is however poorly understood. Barron (1955) has shown oxidation of SH gt'Oups of yeast alcohol dehydrogenase under hyperbaric oxygen conditions and this mechanism of oxygen toxicity has also been suggested by Caldwell (1965 l. Hyperbaric oxygen was shown to prevent reduction of pyridine nucleotides in suspensions of baker's yeast by Chance, Jamieson and Coles (19651. These authors, in an extensive discussion of the possible effects of hyperba.ric oxygen on electron transport and oxidative phosphorylation concluded that SH gt'OUp oxidation probably played an important role. The experiments of Young (1968} with Pseudomortas provide evidence for amino acid transfer as the primary site ofpxygen damage. Oxygen is toxic to Azotobacter at concentrations which do not affect most other microorganisms. Dalton and Postgate (1969) describe continuous culture experiments in which dissolved oxygen values above 30 mM caused inhibition of growth of nitrogen ...limited cultures of'<'Jl-Zotoba.cter. As oxygen approaches inhibitory levels, the oJcy"gen uptake by-,"'5zotobacter increases and ll. sugar consumption rises to a rate greater than that necessary to provide the energy requirement of the cell (Phillips and Johnson, l96ll. These authors suggested that under such conditions, respiration fUnctions as an oxygen-wasting system enabling the maintenance of a low intracellular ~ value, which is presumably necessary for nitrogen fixation. Evidence that a high respiration rate in Azotobacter has the fUnction of protecting the nitrogenase complex from oxidation has been given by Parker and Scutt (19601, Dilworth and Parker (196ll and Khmel, Gabinskaya and Ierusalimsky (l9651. l.3 OZONE 1,3,l Occurrence and Reactions with Organic Material Ozone is one of the most powerful oxidising agents known. It occurs in layers of the upper atmosphere of the earth as a product of solar .radiation of oxygen, In the lower atmosphere it is formed in small concentrations during dissipation of large amounts of energy in thunder storms but can occur in much higher concentrations in the atmospheres of large cities as a photochemical product in smog. Several sequences of reactions leading to its formation in smog have been postulated and these have been discussed recently by Stephens (1969}, The concentration of ozone in smog varies widely and levels of up to 0,65 parts per million (p.p.m.) have been recorded in Los Angeles, California, during severe smog conditions (Hamming, MacBeth and Chass, l967}. A result of such conditions is damage to plant crops and irritation of mucous membranes of human beings. In an extensive review of ozone, Phillips and Hanel (1960} have discussed all aspects of its occurrence, formation and reactions. Ozone reacts with most organic materials and several workers have studied these reactions. Wibaut and Haeyman (19411 found that ozonisation of xylene led to a splitting of the benzene ring with formation of products which could be converted to methyl g]yoxal. Ozone was shown by Haagen-Smi t, Darby, Zaitlin, Hull and Noble (19521 to react with unsaturated hydrocarbons to produce ozonides which caused typical smog damage to plants. The absorption spectra of proteins and amino acids in solution have been shown to be altered by ozone (Giese, Leighton and Bailey, 19521, this effect being most marked where amino acids containing cyclic groups were present. Brinkman and Lamberts (1958) have described o.zone as a radiomimetic gas, its action on organic material resembling th.at of high energy radiation. l. 3.2 Effects on Plants and Animals Dam.age to plant crops is among the most serious effects of photochemical smog. This field has been reviewed by Dugger, Krukol and Palmer, (19661. Various workers have reported effects of ozone on bean leaves (Todd, 19581, tobacco (Heggestad, 1966; Macdowall, 1965) and citrus fruit (Todd, 1956, 1958). In the latter studies on citrus fruit, a stimulation by ozone of respiration was observed. In a study by Hill and co-workers (Hill, Pack, Treshow, Downs and Transtrum, 1961} all of 34 plant species subjected to concentrations of 0.13 to 0.72 p.p.m. of ozone for 2 ..13. hours showed injury symptoms. There was also a general increase in sensitivity with increasing maturity of the tissue. Experiments per-formed by Taylor, Dugger, Cardi-ff and Darley (1.9611 demonstrated an important role of light in the damaging of plant leaves by atmospheric oxidants. They showed by studies in light and dark that there is a photochemical reaction between the oxidant and some intermediate synthesised by light in the plant. Many studies have been made o-r the relation of the dosage of o:zone to the degree of injury produced, There is much evidence for a linear dose/ injury response (Middleton, Kendrick and Darley, 1955; Middleton, l957; Menser, Heggestad and Street, l963; Macdowall, Muka.mmal and Cole, 1964) although Heck, Dunning and Hindwawi (19661 have shown a sigmoidal response of injury to leaves of tobacco and pinto bean to dosage of ozone, They have also shown that the dosage threshold -for injury depends on exposure time up to a period of 4 hours, and that -for concentrations of ozone as high as 60 parts per hundred million, there is a time delay a:rter commencement of exposure be fore injury occurs • Rich and Taylor (19601 have shown that several compounds protect tomato -foliage exposed to atmospheres containing 0.4 to 0.7 p.p.m. of ozone from damage. These antiozonants, which are sprayed onto the -foliage, include 1,2-naphthoquinone-2-oxime and cobaltous and manganous chelates of 8-quinolinol. Mice have been used to study the harmful effects on mammals of excessive exposure to ozone, and the literature contains several reports of --14. adaptation to ozone and of protection against ozone damage by the use of certain types of compounds. After prolonged exposure to sub-lethal concentrations of ozone, mice have been shown to develop a resistance to higher concentrations which were lethal to control animals (Stockinger, Wagner and Wright, l956; Morrow, l964). However other non-lethal toxic actions of ozone were not blocked by this adaptation (Stockinger, Wagner and Dobrogorski, l957) although a cross tolerance to lethal effects of other atmospheric oxidants was demonstrated (Stockinger, l960), Protection of mice against lethal effects of ozone was demonstrated by Fairchild, Murphy and stockinger (l959) using compounds containing SR groups or disulphide linkages. l. 3. 3 Effects on Microor ganisms As long ago as last century, ozone was being used for sterilisation of drinking water (Calmette, Roux, and Staes-Brame, l899l, The disinfection of air containing aerosols of various bacteria was studied by Elford and van den Ende (l9421. Retardation of growth of Bacillus coli by ozone was reported by Haines (l935) who also demonstrated a much greater resistance to ozone by a culture in which growth was well established than when ozone was admitted at the time of inoculation. Watson (l944) showed that the toxic effects of ozone on E. coli and spores of Sclerotinia fructicola were enhanced under acid conditions. The o:xygen uptake by suspensions of baker's yeast in ozonised buffer was studied by Giese and Christensen (l954), They showed an inhibition of o:xygen uptake by ozone and concluded that this was due to death of some of the cells while the oxygen uptake by the viable cells remained unchanged. The same authors concluded that the mechanism of ozone damage to protozoan cells involves disruptive re0,ction at the cell surface before ozone penetrates the cell. Studies by de Koning and Jegier (1968a} showed a stimulation of respiration and an inhibition of photosynthesis when a suspension of the alga, Euglena gracilis was treated with ozone. The percentage reduction of photosynthesis was found to be a logarithmic function of the ozone concentration (de Koning and Jegier, 1968b). Respiration by suspensions of isolated mitochondria has been shown by Macdowall (1965) to be inhibited by ozone. This result is in agreement with that of Freebairn (1957) who also demonstrated a partial reversal of the phenomenon when ascorbic acid or glutathione was added to the mi tochon drial suspension following inhibition of respiration by ozone, 1.4 CONTINUOUS CULTURE OF MICROORGANISMS 1.4.1 Theoretical Considerations The first theoretical treatment of continuous culture was given by Monad (1950) and Novick and Szilard (1950}. Since then, numerous modifications have appeared but the theory presented by Herbert, Elsworth and Telling (1956} has been accepted as quite adequate to describe the cultures studied in this work, Exponential growth of microorganisms may be described by the equation dx = µx, (1) dt where x is the concentration of cells and µ is defined as the specific growth rate, In a batch culture growing exponential]y equation (11 describes ful]y the kinetics of growth. However. in a continuous culture there is. as well as addition of new --16. cells by gr-owth, a loss of cells by dilution with fresh medium. In such a system, if growth was not occurring, the loss of cells by dilution would follow the function dx = (21 dt where D is the dilution rate and is equal to the ratio of the flow rate of medium into the culture vessel to the volume of the culture. Both µ and D have units of reciprocal time, Thus in a continuous culture in which gr-owth is occurring the rate of change of cell mass is given by Increase in Increase due Decrease due = concentration to gr-owth to dilution of cells dx µx D x . . dt = dx or (µ - D) X (3} dt = Under steady state conditions, the concentration of cells in the culture will be constant and dx/dt will equal O. Thus at steady state µ, the specific growth rate will equal D, the dilution rate. Ifµ is gr-eater than D, the concentration of cells in the culture will increase. Ifµ is less than D, the concentration of cells in the culture will decrease, a phenomenon known as ''washout". 17. In a perfect]y mixed continuous culture, the mean residence time of a cell in the culture vessel will be Also the culture which leaves the vessel due to dilution will have exact]y the composition of the mixed culture in the vessel. In his study of bacterial growth in batch culture, Monod (1942) showed the relationship between the specific growth rate and the concen tration of an essential substrate to be µ = (4) where s is the substrate concentration, µ is the growth rate constant m (i.e. the maximum value of µ at saturation levels of substratel and Ks is a saturation constant numerical]y equal to the substrate concentration at which µ = ;,:: µ 2 m· It follows that µ may be held at any value up to µm by keeping constant the substrate concentration. In the technique of continuous culture, this concept of a growth-limiting substrate is of major importance. Where the medium contains a single organic substrate Monod (1942) showed that a simple relationship exists between growth and utilisation of the substrate. The growth rate is a constant fraction, Y, of the substrate utilisation rate. dx ds = - y dt dt C51 dx Y is the yield constant and is equal to ds ' The changes in substrate concentration in a continuous culture are given by the mass balance equation -18. ds µx = dt Y' where ~ is the concentration of substrate in the medium feed. 'Ill.us at steady state conditions, where sis constant andµ= D, X s = y • (6} There are two approaches to the operation of a continuous culture. In the first, the dilution rate is fixed and hence, under steady state conditions, the specific growth rate is controlled. This system is known as a chemostat. In the second system, which is known as a turbidostat, the concentration of cells in the culture is controlled using a turbidometric device which adds diluting medium in response to an increase in x over the desired value. Here the specific growth rate is not under direct control but is known from measurement of the dilution rate. The value of µ will be the maximum possible under the conditions imposed on the culture. 1,4.2 Value of Continuous Culture as a Research Tool During growth of microorganisms in batch culture, the environnent of the cells is continually undergoing changes as substrates are taken up, products of metabolism are excreted and the number of cells increases, This imposes limitations on the interpretation of the behaviour of the organism as a response to a particular environmental factor, The technique of continuous culture however offers the great advantage that the l2, culture may be maintained in exponential growth under steaay state conditions for an indefinite period, It is possible in such a system to impose upon the culture automatic control of many of the environmental factors, Indeed, technical considerations aside, it is theoretically possible to define the total environment of the culture. The controlled values of the environmental factors may then be varied, singly or in combination, thus enabling the effect of specific environmental factors on the organism to be accurately determined, Such a factor may be physical, as, for example, temperature or pressure, it may be the concentration of some substance, or it may be a complex biological f'unction such as growth rate , In the experiments described here, steady state continuous culture has been used widely, but also an extension of the technique has been introduced, This is the imposition of a step change (i ,e, a sudden, permanent change in a controlled environmental parameter) upon a steady state culture and the study of the behaviour of the culture during the period of transition to a new steady state, The advantage of this technique is that whereas in steady state conditions the various systems of metabolic regulation are being exerted to a constant degree, study of the period of transition from one steaay state to another offers the possibility that adjustments in the regulation of the cell's metabolism will be seen as a f'unction of time. This type of kinetic data cannot be obtained from steady state cultures. 20. 2. APPARATUS AND EXPERIMENTAL METH.ODS 2.l THE ORGANISM The organism used throughout these experiments was a camphor- induced strain of the yeast Candida utilis var, major. It was chosen for its vigorous aerobic growth and its capacity to produce high concentrations of cytochrome. The culture, designated Y39, was originally obtained from the British Yeast Type Culture Collection. 2.2 ROUTINE MICROBIOLOGICAL PROCEDURES 2 .2 .1 Maintenance of Cultures Stock cultures of the organism were maintained on slopes of Difeo malt extract agar or on a laboratory prepared medium comprising 3% malt extract, 0 .1% yeast extract and 2% agar, Subcultures were made every three months and after growth at 30°c, were stored at 4°c. 2.2.2 Preparation of Inocula for Continuous Culture Experiments 50 ml of the medium described below in a 250 ml conical flask plug with a cotton wool/was inoculated from a stock culture. This was incubated with shaking at 30°c for 15 hr a:rter which the contents were aseptically added to 500 ml of medium in a 2 1 conical inoculation flask. This was then incubated with shaking for up to 24 hr at 30°c, and was then ready for use as an inoculum for a continuous culture. 2J.. 2.3 CULTURE MEDIUM 2.3.1 Co111.P6Sition of the Culture Medium The medium used in all of the continuous culture experiments, and in the preparation of inocula, was of the following composition. 5.0 X 10-~ -4 6 .25 X 10 M Citric acid 2.0 X 10-3 M HCl 3.0 x l0-5 N CaO 5 .6 x l0-6%Cw/v) r. ) ZnO 2 • 0 X 10-6% o 1...W / V MgO 5,0 x l0-5% (w/v} FeC13 1.1 X 10-5% o (w/v} 6 MnC12 2.8 X 10- % (w/vl cuc½ 5.4xlo-7% (w/v) 7 H3B03 3,0 x 10- % (w/v} Ergosterol l,2 x 10-3% (w/vl Tween 80 2.6 x 10-1% (v/v} a-tocophenyl lactate 6.o x 10-4% (w/v) Ethanol 2,0 x 10-Q.% (v/v) Difeo yeast extract 5.0 x 10-2% (w/v} Di fco peptone 5.0 x 10-2% (w/v} 1.56 % (w/v1 Glucose 2.0 % (w/v1 22, 2.3.2 Preparation of the Culture Medium The components were 1!lixed together in distilled water as follows, and the volume made up to 20 litres. Larger volumes were prepared in multiples of 20 1. l. K¾ P04 136 g. M 2. 4 Na2 H Po4 50 ml. 3. Growth factor 120 ml. 4. Mixed together (l.0M citric acid 40 ml. ( in this order ( + C then added to (Solution A l00 ml. ( bulk of mixture ( + (_ (Solution B l00 ml. 5. Yeast extract (Difcol l0 g. 6, Peptone (Difcol l0 g. 7. (NH 4}2 so4 3ll g. 8. Glucose 400 g. Growth factor was prepared as follows. The components Ergosterol 2 g. Tween 80 400 ml. a-tocopheryl lactate l g. , were dissolved in 95% ethanol with heating and the volume was made up to l 1. with ethanol. 23. Solution A was prepared as follows. The components cone. (10Nl HCl 65 ml. Ca 0 5,6 g. Zn 0 2 g. Fe c13 .6H20 27 g. MnC12 .4H20 5 g. Cu Cl2 ,2H20 o.85 g. H3 B03 0.3 g. were dissolved in distilled water and the volume made up to 5 1. Solution B was prepared as follows, The components Cone. (10N} HCl 225 ml. Mg 0 50.5 g. were dissolved in distilled water and the volume made up to 5 1. The medium has a carbon:nitrogen ratio of 2 and after sterilisation the pH was approximately 4.6. 2 .3.3 Sterilisation of the Culture Medium Medi um for use in preparation of inocula was autoclaved at 15 p.s.i. for 15 minutes in 250 ml and 2 1 conical flasks with cotton wool plugs. Medi um for use in continuous cultures was of too great a volume to be autoclaved conveniently. For this purpose a plate heat exchanger tAlfa Laval} of parallel-counterflow design was employed. Steam at 20 p. s, i. 24. was pass ea. through. one path of the n._ea,t e.xcha,nger while medium was pumped through the other. By this means the medium, as it passed through the heat exchanger, could be rapid]¥ brought to a temperature of 120°c. On the output side of the heat exchanger was a 7 :rt length of l inch diameter industrial pyrex tube, At the end of this was a thermometer and a pressure-regulating valve. The temperature of the niedi um in the holding tube could be kept high by maintaining the pressure developed during passage through the heat exchanger. This was done using the pressure-regulating valve and this procedure also restricted the outflow of medium. Throughout the sterilisation procedure, the pressure in the holding tube was adjusted so that the temperature of the medium at the end of the tube was 120°c. 'Ih.e length of the holding tube had been designed such that at the flow rate of medium thus obtained, it could be safe]¥ assumed that the medium had been held at 120°c for approximately 5 minutes, A:rter leaving the sterilisation apparatus, the medium was passed to a sterile, closed stainless steel drum of 60 1 capacity from which pressure was released through an opening to the outside. The sterile medium was held in this drum for onzy a short time and was then passed through sterile tubing to the sterile, 20 1 reservoirs adjacent to the continuous culture apparatus. At this stage the temperature of the medium was about 8o0 c and it was allowed to cool to room temperature without assistance over a period of some 6 hours . The high-temperature/short-time heat treatment combined with slow cooling and an acid pH resulted in complete sterilisation of the medium A - 611 x 18 11 Pyrex pipeline · B - Cast Iron Backing Flange C - Asbestos insert for Flange D - Neoprene Gasket B E - 5 11 x 31811 Nu_t and Bolt F - Compression Spring 120 lbs at 2° 1 (a) G - Turbidomeler outlet H - Turbidometer inlet Tqrbidometer F J Overflow C D K·- Large Port L - Small Port M - Sampling Point N - Bearing Housing condensate , 0 - Steam Jacket ___Input Gaug• \ - Output Gauge Fishar Filter Regulato, 20 .s,1 Robertshaw Piston Actuator M503-11 Pulley Belt Transducer 543 0 2 Electrode 2000_, Range Change 240V AJ::..Mains I I Fig. 2. 1: (a) The components of the continuous culture vessel. (b) Plan of the feedback loop for control of dissolved oxygen. 25. without mdesirable chemical effects. In cases where contamination of the medium occurred it could always be attributed to a known instance of failure of the apparatus or inadequate aseptic technique. 2.4 CONTINUOUS CULTURE APPARATUS 2, 4.1 The Culture Vessel The apparatus used for continuous culture and the associated control equipment have been described in detail (Moss and Bush, 19671, The culture vessel is similar in general design to that described by Elsworth, Meakin, Pirt and Capell (1956). Figure 2 .l(al is a diagram of the vessel. It consists of an 18 inch length of 6 inch diameter Pyrex pipeline closed at each end with a stainless steel plate TI inches in diameter and ¾ inch thick. The plates are held to the pipe section by bolts and cast iron backing flanges, and the joints are sealed by neoprene rubber gaskets, The top plate carries the housing of the impeller shaft bearings , two large ports to hold electrodes for measurement of dissolved ozygen and pH, and several smaller ports, These smaller ports carry tubes for gas flow (inlet and outletY, thermistor, acid and alkali, medium, inoculum and steam, and a remote jmction tube for the pH reference electrode. The lower end of the stirrer shaf't carries a 2.5 inch diameter -varied disc impeller. 'Ihe base plate carries several ports also, These include the ½ inch diameter overflow tube, a l/8 inch diameter stea.m-jacketted sampling tube, and inlet and outlet tubes for a loop enabling circulation of the culture through the turbidometer, There are also inlet and outlet ports for a cooling coil through which cold water passes during operation of the Fig. 2.2: The culture v essel fully assembled and containing a culture of C. utilis. temperature controller, All of the entry ports to the vessel screw into the end plates and are sealed by teflon washers. The tubes which pass into the culture vessel through the ports are held by threaded cylindrical caps and sealed by neoprene 0-rings of appropriate size. The culture vessel is mounted in a steel frame which facilitates the arrangement of tubing, cables, etc. around it, Figure 2.2 shows the fully assembled culture vessel. 2.4.2 Supply of Medium to the Culture Vessel The sterile medium was held in carboys of 20 1 capacity which were situated adjacent to the culture vessel. A line consisting of sections of glass tubing and flexible silicone rubber tubing carried the medium to the culture vessel. 'Ihe flow was mediated by a peristaltic pump (Sigmamotor, Model T8} operating on a section of the ill cone rubber tubing. The medium was introduced to the culture below the surface through a 3/16 inch stainless steel tube. The medium pump could be operated independently, or in conj1mction with the turbidity controller. 2,4,3 Collection of Cells and Products 'Ihe volmre of the culture is maintained at a constant level by a simple weir, consisting of a½ inch stainless steel tube which passes through the base plate. Mixed culture overflows through this tube at the rate at which sterile medium is added to the culture vessel. 'Ihe tube passes to sterile collection vessels immersed in a 4°c water bath below the culture vessel. These harvest vessels are fitted with air filters to allow escape Fig. 2. 3: The culture veooel m ounted in the steel framewo rk above the sterile colle ction ::;ystem. The colle cting vessel s are immersed in a bath of water at 4°c. 27. of displaced air. A glass Y-piece enabled the overflow culture to be diverted to either of the harvest vessels and each could be aseptically replaced by a similar sterile vessel as required. Figure 2 .3 shows the fully assembled culture vessel and collection system. 2 .4 .4 Gas Flow The gas flow into the culture was a mixture of air with either oJcy"gen or nitrogen. Laboratory compressed air was passed through a non-sterile glass wool filter to a pressure regulator (Negretti and Zambra, -12 to +65 p .s .i. l thence to a flow regulator (Flostat Minor MK2). The controlled stream of air then passed through a flow meter and, if required, was mixed with a supplement of oJcy"gen or nitrogen which had been similarly controlled and metered. The gas mixture was then passed through a sterile packed cotton wool bacterial filter and into the culture vessel. It entered the culture through. several fine holes in the end of a stainless steel tube beneath the impeller. Effluent gas was passed from the culture through. a 3/8 inch diameter stainless steel tube in the top plate. A water-jacketted condenser was fitted to the end of this tube the function of which was to minimise loss of water from the culture by evaporation. The effluent gas was then passed through. a bacterial filter (Mackley) and on to the analysis system. When passage of ozone into the culture was desired, the oJcy"gen supplement was passed through an ozone generator (Section 2.6} and part of the OJ 2.4.5 Measurement of Oxygen Tension Oxygen tension in the culture was measured using a Mackereth oxygen electrode (Electronic Industries Ltd,, No, A15A). The principle and characteristics of this electrode have been described (Mackereth, 1964}. The electrode produces a current of approximately 350 microamperes (µA} in air saturated water at 30°c and 760 mm Hg. This is equivalent to a concentration of oxygen of 235 x 10-6 molar. A residual current of less than l µA was obtained at 30°c in boiled water flushed with oxygen-free nitrogen and containing sodium sulphite. The response of the electrode between these two readings is linear and a calibration curve was constructed by joining these two reference points by a straight line, The air saturation and residual currents of the electrode were determined before each period of use and the oxygen tension in the culture was expressed in terms of concen tration (µM}, any small variations from atmospheric pressure in the culture headspace being neglected. The electrode was fitted to the culture vessel by removing the water proof cap and replacing it with a 15 inch length of 3/4 inch stainless steel tube, turned so that the electrode body fitted into it and sealed against a neoprene 0-ring, The cable of the electrode passed down the middle of the extension tube, which passed through a port in the top plate of the culture vessel. 29, 2.4.6 Control of Oygen Tension The oxygen tension in the culture was controlled by varying r~te the oxygen solution/through variations in the stirrer speed, The successful application of this principle to control dissolved oxygen in a microbial culture depends on the use of a sensitive oxygen electrode, a reasonably constant rate of oxygen uptake by the culture, and an automatic controller capable of very fine adjustments to the stirrer speed. A diagram of the oxygen tension control loop is shown in Figure 2.1(b). The current produced by the Mack.ereth electrode is fed to a range change switch and converted to a Oto 5 millivolt signal which is in turn fed to a 3-term electronic controller (DeVa.r, model C413 EL). The controller feeds the signal to a single channel cha.rt recorder (DeVa.r, model R321R-PO 50) Depending on the variation of the recorded oxygen tension from the set point, the controller produces an output signal, the value of which determines the stirrer speed in the culture vessel as follows. An electropneumatic transducer (Fisher, model 543) converts the 1 to 5 milJiampere controller output signal to a 3 to 15 p ,s .i. pneumatic signal. The value of this pneumatic signal determines the position of the connecting shaft of a piston actuator (Robertshaw, model M503-lll. This actuator operates the speed control arm of a variable speed drive (Zeromax, No, 25JCCW), which is mounted directly onto a ¾ H,P. 1440 r~. electric motor. Pulleys and a belt transfer the drive from the gearbox to the stirrer shaft of the culture vessel. The length of the gearbox control arm and the point of its attachment to the shaft of the piston actuator were adjusted to provide a suitable range of 30. stirrer speeds over the range of the controller output, The range of stirrer speeds employed was approximately 100 r,p,m. to 800 r.p.m. for 0% to 100% of the controller output. 2.4.7, Control of Temperature Temperature was controlled by a two wey on-off controller consisting of a transistorised amplifier reley (Honeywell, model R7081C). The temperature sensor is a thermistor which fits into a closed stainless steel tube immersed in the culture through the top plate of the culture vessel, The output of the controller is diverted to either a 250 watt infra-red heating lamp mounted 6 inches from the culture vessel, or to a solenoid valve which, when open, permits the flow of cold water through the cooling coil. The diversion of the controller output to the lamp or cooling coil depends on whether the temperature of the culture is below or above the set point respectively. The control circuit was such that one of the control functions was alweys operating, This system enabled the temperature of the culture to be controlled within± 0.2°c, 2,4.8 Control of pH The pH control system was similar to that described by Callow and Pirt (1956 l. The pH of the culture was measured using a pH meter (E.I .L. Model 23Al with a glass electrode and a ca.lomel reference electrode, The glass electrode was fitted into a glass extension tube to which the S.ides of the electrode were sealed by tightly fitted neoprene 0-rings and cold cure 31. silastomer (Midland Silicones, N9l59l. The extension tube passed through. the top plate of the culture vessel. Contact of the calomel reference electrode with the culture was effected by an extended salt bridge passing through.thetop plate of the culture vessel. This consisted of a piece of glass tubing with a ceramic plug at the lower end, It was filled with saturated KCl and connected by flexible tubing to a small reservoir of saturated KCl in which the reference electrode was bathed, The signal from the pH meter was fed to a recorder controller (Honeywell electronic single record strip chart constant controller, or Ether Xactrol single channel recorder-controller Type 2007/15}, In both of these instruments, two We¥ on-off control was available. The controller output activated peristaltic pumps which added either acid (2N H2S04) or alkali l2N Na0H} to the culture depending on the direction of deviance of the pH from the set point. In practice it was found that C. utilis produced acid during growth such that only alkali was added to maintain the desired pH. 'lhis system enabled pH to be controlled to within± 0.05 of a unit. 2,4.9. Control of Cell Density The turbidity of the culture was measured and/or controlled with a cell and control unit as described by Moss and Bush (1967}. The action of the stirrer draws the culture through. the turbidometer circulation loop (see Figure 2 .l(al) which passes through the turbidometer cell. Here a light source shines through. the ¾ inch diaJrJeter glass tube carrying the Fig . 2 . 4: The c omp l ete co ntinu ous c ulture apparatus . 32, culture and a photocell produces a signal according to the level of transmitted light. The signal is read on an ammeter and indicates the turbidity of the culture, The ammeter is connected to an on-off controller which permits operation of the continuous culture as a turbidostat if · , required. When operating in this way, the medium. pump is linked to the turbidity controller and operates only when the turbidity exceeds the set value, Figure 2, 4 shows the entire continuous culture apparatus including culture vessel, medium. reservoirs, collection system and control equipment. 2,4.lO Sterilisation of the Continuous Culture Apparatus The lines of tubing leading from the culture vessel to the air filters ,medium reservoirs and harvest vessels are able to be broken by means of a sterilisable, stainless steel connecting device of the type •. described and illustrated by Moss and Bush. .. (19671. Before sterilising the culture vessel the air filters, medium reservoirs and harvest vessels were removed and autoclaved separately at 15 p,s .i. for 15 min. The oxygen and pH electrodes were sterilised by immersion in 8% formaldehyde (20% v/v commercial formalin} for 20 min, The culture vessel and attached tubing was sterilised by passage of steam from the laboratory supply. For this procedure, loose stainless steel covers were placed over the ports in the top plate for the oxygen and pH electrodes, The salt bridge and the closed tube which carries the thermistor remained in position during sterilisation of the culture vessel, During passage of steam through the vessel, care was taken to ensure that it flowed 33, freely through each line and out all openings for at least 2 hours. When steaming of the vessel had proceeded for the required time, the flow of steam was reduced and the sterile air filters, medium reservoirs and harvest vessels were aseptically connected. Each connection was made while a small flow of steam passed out of the tube from the culture vessel and inmediately af'ter the join was made the tube was closed by a screw clamp, When all of these connections had been made, there remained a small flow of steam from the two covered electrode ports in the top plate. This steam flow was then gradually replaced by a flow of sterile air which entered the vessel through the gas inflow system. The vessel was lef't in this conai.tion for about½ hour in order to cool. When it had done so, the oxygen and pH electrodes were removed from their sterilising solutions, quickly passed through the entry ports in the top plate and sealed into position. The air flow was turned off and the completely assembled, sterile apparatus was ready for,bperation. 2.4.ll Operation of the Continuous Culture Apparatus 2,4.ll~ l The Settine; ·:si of a Continuous Culture Following sterilisation of the apparatus, the medium reservoirs were filled with sterile medium. Medium was pumped into the culture vessel almost to thEf-1-evel of the working volume, and the extremity of the inoculation line was sterilised by the passage of steam for several hours. During this period the stirrer and the pH and temperature controllers were set in operation. 34. When sterilised, the inoculation line was aseptically connected to the tubing from the inoculation flask and the inoculum was run into the culture vessel. The culture was allowed to grow until a suitable cell density had been attained and continuous dilution was then col!IIDenced. The medium flow rate was fixed if the culture was being operated as a chemostat. During operation as a turbidostat the flow of m.edi um into the vessel was intermittent in response to the operation of the turbidometer. In the latter case, the flow setting of the medium pump was adjusted such that during each period of operation of the pump, the turbidity was reduced just to the set point and not below it. This resulted in an even flow of medium, the pump operating at very frequent intervals. As a steady state was attained, control of dissolved o:xygen was imposed (if desired). The gas flows and stirrer speed were first adjusted manually until a dissolved o:xygen value approximating the desired value was obtained. The controller was then switched to automatic operation and fine adjustments made to the gas flows, particularly with regard to the concentration of oxygen in the inflowing gas. These adjustments were continued until a situation was reached where, in controlling dissolved oJCy-gen at the desired value, the stirrer speed underwent only slight variations and, while sufficient to provide adequate mixing of the culture, was not excessive. 35. 2. 4,ll.2 Sanwling Procedures During operation under steady state conditions, samples and readings were frequently taken in order to measure the desired para.meters. Where studies were made of transient states, samples and readings of different para.meters were taken as closely together as possible at each sampling, so that where changes with time were occurring simultaneous measurement could be assumed. This enabled valid expression of rates of uptake of substrate and production of metabolites relative to dry weight. The :measurement of cytochrome concentration in the cells (Section 2.5,1 required a sample of several g, wet weight of cells. For this a sample volume of at least lOO ml was required and this could not be drawn directly from the culture without causing a major disturbance to the growth conditions For this,reason the overflow from the culture collected over a period of ½ to l hour was used. The figure obtained for cytochrome concentration does not -then represent a value at any instant, but is the average value over the period of collection of the sample. The culture overflow was collected in a sterile flask immersed in a water bath at 4°c. When sufficient sample had been collected, the flask was aseptically replaced by a new sterile flask. After removal from the continuous culture apparatus the cytochrome sample was kept cold but was not handled aseptically. Measurements of cell dry weight and concentration of glucose in the culture supernatant were made on the same sample taken from the steam jacketted sampling tube in the base plate of the culture vessel. The culture was run from the tube at a slow rate to ensure heat inactivation of the cells 36, in the sample and cessation of their metabolism. This eliminates the likelihood of continued uptake of glucose by the cells during centrifugation which mey result in a falsely low reading for the glucose concentration. After discarding the first few ml from the sampling tube, 15 ml was collected, This was dispensed and used for measurement of dry weight (section 2.5 .21. The supernatant obtained from the first centrifugation of the sample was set aside for measurement of glucose concentration (section 2,5,3}. Glucose concentration was measured illl!Ilediately or the sample of culture supernatant was stored at -25°c for a period of up to two weeks. The concentration of ethanol in the culture supernatant and the percentage viability of the culture were measured in samples taken from the turbidometer circulation loop (see Figure 2 .l(a)). This sampling tube is fitted with a steam supply and so mey be sterilised before and after sampling, In collecting a sample, the first few ml were discarded. 5 ml of the culture was then collected and the cells were removed by rapid centrifugation (2000g for 3 min. } • The concentration of ethanol in the supernatant was then determined immediately or the sample of supernatant was stored in a tightly closed Maccartney bottle at -25°c for up to 2 weeks. Each dey during operation, the culture was examined microscopically and an aseptically collected sample from the turbidometer loop was plated onto malt agar and nutrient agar (Oxoidl in order to detect the presence of any contaminating organism. 37, 2.5 .ANALYSIS OF THE CULTURE 2 .5 .l · Measure:rrent of Cytochrome Concentration 2,5.l.l Reflectance Spectroscopy The sample, which had been collected at 4°c (see section 2. 4.ll.21 was subjected to centrifugation at 2,000 x g for 5 min. The paste of cells so obtained was then washed twice with cold M/15 phosphate buffer (S95rensen, 1912) at pH 7.0, A few mg. of crystalline sodium dithionite were then mixed thoroughly into the paste which was allowed to stand for a few minutes in order to effect complete reduction of the cytochromes • The circular recess (4. 5 cm di a.meter x 4 mm deep} of an aluminium. reflectance cuvette was then filled with the reduced yeast paste and covered firmly with a clean glass plate (5 x 5 x 0.05 cm), care being taken to exclude air bubbles. A Perkin Elmer Model 350 double beam spectrophotometer fitted with reflectance head was used to measure the difference in reflectance between the sample and the reference material, which was filter paper. The instrument was used in the visible range (350 to 750 millimicrons} with the slit width set to O.l mm at 650 mµ, the response time at 3 and the scan speed at l. A typical reflectance spectrum. obtained by this method is shown in Figure 2.5, in which the a absorption maxima due to the cytochromes are labelled. " the 750 calibration 7.0 and cytochromes. the 1,50 s:,.own to cytochrome. cytocn.rome as 600 due T:,.e 575 each dravm for 550 i::; maxima !l,U value measured. !,00 is absorption absorption 475 • the maxima concentration (lllllllmk,...e) '------~- a ... shovli.ng non-specific of WAVELENOTH obtain ,. utilis . to line oc.-abaorption C. used of foe of are - 6) backgrou:id eac:i n, 2. spectrum for 380 (Fig. 1/IB arbitrary no ratio curves T:-1e Reflectance )60 5: 2. 350 0 90 70 .., 60 50 JO 10 40 zo 100 . Fig. z ~ u IC ~ ,., .. g lj .. = 38, 2.5,l,2 Calculation of Cytochrome Concentration A value for concentration of cytochromes A, B and C, expressed as the percentage of an arbitrary standard, was determined. To do this, an arbitrary background line was drawn as a tangent joining the absorption mini:ma at approximately 477 111µ and 650 mµ, as in Figure 2.5, and the ratio I/IB was determined for each of the a. absorption maxima. The calibration curves shown in Fig. 2 .6 were used to obtain values for the concentration of each of the cytochromes. These calibration curves had been constructed by Dr. P,A.D. Rickard in the School of Biological Technology, The University of New South Wales, as follows. A culture of C. utilis showing a very high cytochrome level and a cytochromeless sample of the same yeast, produced by treatment with ozone (see section 3.4.l} were mixed in graded proportions. For each mixture, the I/IB values from the reflectance spectrum were plotted against the proportion of cytochrome rich yeast. The cytochrome rich yeast was defined as having a cytochrome concentration of 100%, and its proportion in each mixture represented the relative cytochrome concentration of that mixture. Mok (1968) has shown that cytochrome concentration in duplicate samples may be estimated by this method to within ± 5%. 2,5.2 Measurement of Cell Dry Weight of the Culture A 15 to 20 ml sample volume was collected (see section 2. 4,ll.2) and 5 ml aliquots were immediately pipetted into three tared 3 inch x ½ inch glass tubes. The cells were separated by centrifugation and the supernatant retained for estimation of glucose concentration (section 2.5.3}. A:f'ter washing twice with distilled water, the dry weight samples were le:f't at 100°c 1,0 CYTOCHROME A .. , r.I 0,1 0,7 o., 0 10 zo 30 40 50 70 10 90 100 1,0 CYTOCHROME 8 o., I 0,1 \I •• 0,7 o., 0 10 zo 30 / 40 so 60 70 IO 90 100 I / .. 1,0 CYTOCHROME C 0,9 I •• 0,1 0,7 o., 0 10 zo 30 40 so 60 TO 10 90 CYTOCHROME CONCENTRATION (PERCENT OF MAXIMUM) Fis• z. 6: Cyt~chrome calibratio:..1 curves. The preparation of these curves is described in Section z. 5.1. 2. 32. for 48 hours. The tubes were then weighed and from the mean of the three replicates the dry weight value of the culture was calculated and expressed as g. /1. 2.5.3 Measurement of Glucose Concentration 'Ille concentration of glucose was determined in the supernatant of the sample of culture taken for dry weight estimation. The method used was a modification of the glucose oxidase method of Huggett and Nixon (J.957J which was considered to be highly speci fie for glucose. Materials Commercial preparations of glucose oxidase (from Penicillium notatum} and peroxidase (from horseradish} were supplied by C.F, Boehringer and Sohne, Mannheim, Germany. £_-dianisidine (laboratory reagent) was obtained from Drug Houses of Australia and sodium dihydrogen phosphate (NaJI2Po4 .2~0) of analytical grade was supplied by Hopkin and Williams Ltd., England, The glucose for preparation of standards was obtained from May and Baker, England, Preparation of Enzyme Reagents 0. 5 M NaJI2P04 was adjusted to pH 7 ,0 with N/1. Na0H. J.25 mg. of glucose oxidase and 0.5 mg. of peroxidase were dissolved in 100 ml of the 0,5 M N~Po4 solution. 0.5 ml of .1.% o-dianisidine (in 95% ethanol} was added and the mixture filtered through. Wh.atman No.J. filter paper. This reagent remained active for 7 days when stored at 4°c. 40. Method Samples were diluted to within the range of 250 to 500 µg. of glucose per ml. Duplicate aliquots of 0.l ml of the diluted sample were pipetted into 15 :x 2 .5 cm test tubes. 2 .5 ml oi' the enzyme reagent was added to each and the mixtures incubated for 45 min. in a 37°c waterbath. A reagent blank containing 0.l ml of distilled water instead of a sample, and standards of 250 and 500 µg of glucose per ml were also set up. The rea.ction was stopped by the addition of 2.5 ml of l2N ¾so4, 'Ihe optical density of each solution was read against the blank at 540 mµ using a Hitachi Perkin-Elmer Model 139 spectrophotometer, A curve of glucose concentration against optical density was constructed from the standards and this was used to determine the glucose concentration in the sample. 2,5.4 Measurement of Ethanol Concentration The concentration of ethanol in the sample of culture supernatant (collected as in section 2,4.TI,21 was determined by gas chromatography. A Beckman G. c.4. gas chromatograph with hydrogen flame detector was used. The column was a 150 cm length of 3,12 mm (inte?tlal diameterl stainless steel tubing packed with a polymer of ethylvinylbenzene di vinyl benzene (Porapak Q, 80-100 11'.leSh, Walters Associates Inc. Framingham, Mass,, U.S,A.}. The carrier gas was nitrogen at a flow of 15 ml/min and this was mixed with a flow of 250 ml of air/min at the detector. The system was operated under the following temperature conditions: 41. Inlet, 230°c Inlet line, 210°c Column, 200°C Detector, 24o0 c. The detector signal was recorded on a Speedomax: H recorder fi. tted with integrator and at a chart speed of 30 inches/hr. A sample of volume l µl was injected into the column and a:rter a retention time of l,38 min, a peak due to ethanol was recorded. The area rmder the peak was measured by the integrator and was proportional to the concentration of ethanol in the injected sample, at a constant injection volume. Using a 1% (v/v) aqueous ethanol standa.rq the concentration of ethanol in the sample was calculated by comparing the area under the peak of the sample to that of the standard. 2,5 ,5 · Measurement of the Viability of the Culture The determination of viability was based on the phenomenon of quantitative absorption of methylene .blue by dead yeast cells as discussed by Borzani and Vairo (1958}. 'Ihe method used was that of Vairo (1962) and the following procedure was adopted. An aqueous solution containing 200 mg of methylene blue (Gurr) 27 .2 g of ~Po4 and on 71 mg of Na2HPo4 per litre was prepared, Serial dilutions of this solution were made and a curve of optical density at 440 mµ against methylene blue concentration was constructed using a Hitachi Perkin Elmer model 139 spectrophotometer. 42. A 5 1lll sample of the culture was collected (see section 2,4.ll,2). The cells were separated by centrifugation, washed in distilled water and then suspended in 25 ml of fresh distilled water. The suspension was halved and the cells in one half were killed by boiling for one minute. For each of the fresh and boiled suspensiom, duplicate aliquots of 6 ml were pipetted into 2 x 3/4 inch Maccartney bottles together with 4 ml of the methylene blue solution. A blank with 6 ml of distilled water substituted for the yeast suspension was .also set up, The Maccartney bottles were tightly closed and agitated on a rotary mixer for 20 min. at room temperature. The cells were then separated from each test suspension by centrifugation and the concentra tion of methylene blue in each supernatant was determined colo.tmetrical]y, using the calibration curve described above. The percentage viability was calculated as follows. ci - cf p = X 100 ci - cf(100) where P percentage of dead cells Ci = dye concentration in distilled water blank Cf = dye concentration in supernatant from unboiled sample Cf(lOO) = dye concentration in supernatant from boiled sample (where P = 100}, The percentage viability was then 100 - P. Although the aceuracy of this method was no greater than ± 10%, it 43, was preferred to the plate count method because of its relative simplicity and the much shorter time required to obtain the final result. 2.5.6 Gas Analysis 2.5.6.1 Measurement of Oygen and Carbon Dioxide Gas flow through the culture vessel is described in section 2 .4,4. The concentration of oxygen in the inflowing gas is calculated from the flow rates and the known composition of the gases used. The concentration of carbon dioxide in the inflowing gas was always O. The concentration of o:xygen in the effluent gas was measured directly by either continuous passage through a Beckman (model E2) pa.1 a.magnetic oxygen analyser or by collection of a 100 ml sample ·in an Orsat apparatus and measurement of the residual gas volume after absorption of oxygen by aJkaline pyrogallol. The concentration of carbon dioxide in the effluent gas was measured directly by either continuous passage through an infrared co2 analyser (Lira Model 300, M.S.A., Glasgow}, or by the Orsat apparatus in conjunction with the measurement of oxygen concentration, the residual gas volume being measured a:rter absorption of carbon dioxide by potassium hydroxide. 2,5.6.2 Measurement of Ozone The method for measurement of the concentration of ozone in the inflowing gas was based on thef.3tarch iodine method originally described by Paneth and Edgar (1938) and modified by Crabtree and Kemp (1946). The 44. inflowing gas was passed through a sealed flask of exactly l ,2 1 capacity for some hours until the original gas had beer. completely displaced. The gas flow was then diverted through a bypass and the gas sample in the flask was allowed to reach atmospheric pressure by bubbling off excess gas through a water trap, the flask being again closed immediately the bubbling had ceased, A glass syringe was fitted with a hypodermic needle and 10 ml of a freshly prepared potassium iodide solution (5 g in 25 ml of phosphate buffer, pH 7,0l was drawn into it, The potassium. iodide was then injected into the flask by piercing a rubber diaphragm fitted across a side-arm. The flask was then shaken vigorously to allow couplete reaction of the ozone with the potassium iodide to form iodine, After shaking for several minutes, the flask WE.S opened and the iodine which had been produced was titrated with 0,01N sodium thiosulphate. "Thiodene" indicator was used in preference to starch since it gave a sharper end point. l ml of 0.01N Na2s2o3 is then equivalent to 0. 1l2 ml of ozone at standard temperature and pressure. Thus the concentration of ozone in the inflowing gas was calculi:.ted and expressed as parts per million (p.p.m.) or percent by volume, 2.5, 7 Expression of Metabolic Para.meters of the Culture The rates of uptake of substrates and production of metabolites by the culture were calculated and expressed as millimoles per gram dry weight per hour, The various rates were designated Qo2 , QG, Qc0 , QE (see 2 Glossary for definitions) and the equations used in their calculation are given in Appendix I, 45, 2.6 GENERATION OF OZONE Ozone was produced by ultraviolet irradiation of oxygen, The ozone generator consisted of a 24 inch ultraviolet tube (Type 24 HIQ, Oliphant, Adelaide) housed in a closed 4 inch diameter copper cylinder through which the oxygen stream was passed, The ultraviolet tube was fitted with a cylindrical brass shield which was divided longitudinally and could be opened to -varying degrees. This enabled variation of the degree of radiation to which the gas passing through the ozone generator was subjected, By adjusting the position of the shield in combination with the gas flow rate, the concentration of ozone in the oxygen stream could bejvaried from 0 to 1000 p,p,m, (0,l %v/v). 2,7 ELECTRON MICROSCOPY The method of preparation of the cells for examination by electron microscopy was based on that used by Linnane, Vi tols and Nowland (1962). A 10 ml sample of the culture was collected and the cells washed with distilled water, The cells were then suspended in 2% (w /v} aqueous potassium permanganate and le ft at room temperature for 2 hours, After washing several times with. water the cells were suspended in a solution containing l % (w /v) potassium di chromate and l % (w/vl uranyl nitrate, and again left at room temperature for one hour, After washing several times with water until the di chromate colour had disappeared from the supernatant, the cells were dehydrated by washing through a graded series of ethanol solutions (30%, 50%, 46. 60%, 70%, 80%, 90%, 95%, lOO%J. The final dehydration step was to wash the cells twice in superdriel1. absolute ethanol (dried by storage with molecular sieve (type 5A, Union Carbide) for several days). The cells were then suspended in a solution of equal parts of superdried ethanol and the Araldite epoxy resin mixture described below, and le:rt overnight. Following this the cells were separated by centrifugation and suspended in the Araldite;. epoxy resin mixture to ensure complete infiltration. After 24 hours, the cells were embedded in the Araldite mixture in gelatin capsules and the resin was polylilerised by heating at 6o0 c for 48 hours. The Araldite epoxy resin wes supplied by CIBA Pty Ltd., and the mixture used was as follows. Casting Resin M 10 ml Hardener 964B 10 ml Accelerator 964c o.4 m1 The embedded samples were sectioned with a Porter-Blum M'l'l ultra microtome using glass knives. Sections showing silver or gold interference colours were floated onto nitrocellulose and carbon t.:oated copper grids (Athene, "New 20011 ) and examined in a JEM Model 6B electron microscope operating at 60 KV. 47. 3. EXPERDdENT.AL 'RESULTS 3.1 GROWTK IN STEADY STATE CONTINUOUS CULTURE AT VARIOUS CONTROLLED LEVELS OF DISSOLVED OXYGEN The response of C. utilis to various controlled levels of dissolved oxygen was studied in a chemostat culture, In a culture of volume 3 1, the dilution rate was set at 0,l hr-J.., the temperature controlled at 30°c and the pK at 5,0. In a series of steafl.y states, the dissolved oxygen was controlled at -various levels ranging f'rom 400 1:1M down to the 1:bni.t of measurement with the Mackereth electrode, At the lower extreme of' the range covered, two steafl.y states were studied in which dissolved oxygen was below the li:mit of 10.easurement, In these two states, a fixed air f'low- rate and fixed stirrer speeds replaced automatic control of dissolved oxygen, Also, the · culture was operated as a turbidostat in these states and dilution rates of 0.12 hr-1 · and 0.05 hr-1 · were recorded Gsee Table 3.1 l . At each steafl.y state measurements were -made of the culture ary- weight, cytochrome concentration, Q02 , QC02 , %• ~ and the rate of acid production. The data obtained from this stuay are shown in Table 3.l and Figs, 3,1 1 3.2 and 3,3, Table 3.2 shows -various indices of metabolic activity, calculated from the data in Table 3,l. The usei'ulness of these indices is- in facilitatin comparis·on between different steafl.y states in regard to the distribution of glucose to the catabolic pathw-a;y-s operating, and the efficiency with which the cells axe utilising the substrates. Acid Cytochrome Dissolved Dry .Analysis of QG Qo Qco ~ 0Jcy"gen Weight Culture 2 2 Production Concentration (µM} (g/1) Supernatant ml N NaOH/ (% of maximum) g.dry wt/hr Glucose Ethanol (mmoles/g,dry wt/hr} A B C (% w/vl C%v/vl 400 8,4 0 0 0,84 53,20 4,20 0 0,74 33,0 44.o 46.o 235 ll,9 0 0 0.93 12,55 2,10 0 0.94 41,5 44.5 50,0 200 14.2 0 0 0,75 0 0,56 44,5 50.0 47.5 100 12,7 0 0 0,87 0 0.60 44,o 50,0 43.0 50 12,l 0 0 0.91 4,00 2,65 0 0,58 46,5 50.0 47.0 20 12.0 0,0l 0 0,97 3.90 4.03 0 0.77 34.5 39.0 35 .o 10 ll.8 0 0 1,28 4,47 3,25 0 0.80 35 ,5 37,5 35 .5 3 16.1 0 0 0,79 3.20 2,80 0 0,55 44.5 31,0 38.5 1.5 15,2 0 3,46 4,05 0 0,60 42 ,0 35.5 34.5 0.15 14.9 0 0.03 0.92 3,60 2,81 0.27 0.61 64.5 46.0 50.5 sl 8.2 O,Ol 0,04 2,05 4,42 5 .89 0,25 o,68 71,0 63.0 61.0 s2 6,5 0 0,03 1,00 l,44 3.06 0,80 0.63 64,5 59.5 54.o Table 3,1; Data from growth of C, utilis in steady state continuous culture at various controlled levels of dissolved oJcy"gen, In states s1 and s2, dissolved oxygen was i11lilleasurably low and was not controlled but the air flow rate was fixed at 300 ml/min and the stirrer speed at 800 r.p.m. for s1 and 600 r.p.m. for s2. Through.out, the temperature was controlled at 30° and pH at 5,0. Dilution rate was maintained at 0.l hr-1 except for s1 and s2 in which the culture was operated as a turbidostat and where dilution rates of 0.12 hr-1 for s1 and 0,05 hr-1 for s2 were recorded. Values shown for all parameters are averages of from three to ten readings taken while steady state conditions were maintained, -I="" .co 55 50 0 aoz • aco2 C 9o I I I t I k ...... 1:1 zo i ~ k • 'tl • ...... bO 15 ID • ....Q) 0a s 10 . 5 .. 0 1 10 DISSOLVED OXYGEN {pM) Fig. 3. 1: Plots of 0oz, Ocoz and Oo against controlled steady state dissolved oxygen value. From data given in Table 3. 1. a1 an~ a2 aa in Table 3. 1. .. -', 1-4 ...... d 1. 0 (a)' i ~ "O • 0.5 .....tlO •u 'o a 0 •l s 8z'110 1 10 102 103 20 (b) • u 15 ...1-4 .....::; l 10 t- 'U ..• • 5 ... - DISSOLVED OXYGEN (JlM) . Fig, 3. 2: Plot• of (a) OE and (b) dry weight againat controlled steady state diaaolved oxygen value. _From data 1iven ln Table 3. 1. i 1 and a2 aa-ln Table 3. 1 • , .. ... 10 CY TC><;HROME A f>O 40 20 0 -1 i 'z•1 10 10 102 103 :, ~ 80 x CYTOCHROME B ~ 0 "' 60 ~ z 0 ~ ~ 40 f- 60 40 zo DISSOLVED OXYGEN (pM) Fig. 3. 3: Plots of concentrations of cytochromes against steady· state dissolved oxygen value. From data given in Table 3. 1. s 1 and s 2 as in Table 3. 1. Cytochrome Concentration and Oxygen Uptake The values for cytochrome concentration did not change significantly over a range of dissolved oxygen values from 235 l,IM to 1. 5 µM. At 400 µM there was a slight decrease in cytochrome A,but not in cytochromes B and C. At dissolved oxygen -vaJ.ues below 1.5 µM, however, cytochromes A, B and C showed a 50 to 75% increase.· ()Jcy'gen uptake was independent of dissol-ved oxygen between 0.15 µM and 50 µM. At the lower end of the range studied, the value of % at s1 2 approximated that at 0.15 µM but at s2 a decrease in Qo2 from 4.42 to 1.44 m moles/g./hr was recorded. At 235 µM and 400 µM, greatly increased Qo2 values of 12.55 and 53.20 m moles/g./hr respectively were recorded. These high % values were not associated with increased cytochrome levels or QG 2 values, but the dry weight values at these levels of dissolved oxygen showed a slight fall. It is noteworthy that in order to maintain a dissolved oxygen value of 400 llM, a very high gas flow rate of l ,100 ml/min. of oxygen plus 100 ml/min. of air was necessary, the stirrer speed being approximately 600 r.p.m. The relationship between Qo2 and growth is shown in Table 3.2, column 5. Assuming an overall P:0 ratio of 3 and using the Beaucho,p and Elsden (1960) value for YATP of 10 g. dry weight of cells per mole of ATP produced, a theoretical value forµ can be calculated as follows. 1 2 3 4 5 6 7 8 9 10 Dissolved Q Q - ~ Carbon Qo Co2 -~. ~ ~ µ(calcl .~ . Co2 .. 0Jcygen Ye Balance 2 (µM} -- :J.-- - (sum of Qo µ(exp. 2 QG 6 QG QG 2 QC02 QC02 r 6,7,8) 400 0,08 0 1,0 31.9 0,72 0 0,83 :J.,62 63,5 235 0,17 0 l.O 7,2 0.70 0 0.36 1,06 13,5 200 - 0 :J.. 0 - 0,90 0 100 - 0 1,0 - 0,77 0 50 0,70 0 1,0 2,4 0,73 0 0,70 l,43 4.1 20 0,10 0 1,0 2,3 0,68 0 0,68 1.36 4.0 10 o. 70 0 1,0 2,7 0,52 0 o.43 0,95 3,5 3 0,90 0 1,0 1,9 0,85 0 0.60 1.45 4.o l,5 1,17 0 1,0 2,1 0,15 0,70 0,10 0,90 2,2 0,73 0,13 o,47 1,33 3,9 sl 1.30 0,04 0,96 2,7 0,33 0,06 o.48 0,87 2,2 s2 1,60 0,26 0,74 1,0 0,24 o.36 O,ll o.n 1.2 Table 3,2: Indices of metabolic parameters of C, utilis grown in steady state continuous culture at various controlled dissolved oxygen values, Calculated from data given in Table 3,1, VJ 0 5l. 11 (calcl = l000 'Ihis gives the value for µ which would be expected if all of the oxygen taken up was being utilised for oxidative phosphorylation. Only at steady state s 2 was this ideal realised, the -value of µ(calc l being consistently greater than the experimental value at higher levels of dissolved oxygen, At the 235 µM and 400 µM steady states, the ratio of Jl( becomes ea1 c }/µG exp, ) very much higher and very little of th¥>xygen taken up can be accounted for by oxidative phosphorylation. Table 3. 2, column 2, shows values for a respiratory quotient. co2 produced in fermentation has been subtracted. At intermediate values of dissolved oxygen, the respiratory quotient approximates l, but is greater than l at low dissolved oxygen -values, indicating the production of co2 by a non-respiratory~ non-ethanolic pathway such as the pentose phosphate pathway. At 235 µM and 400 µM, the respiratory quotient is very much smaller than 1, reflecting the large proportion of Q02 which is not involved with oxidative phosphorylation. Glucose Uptake and Carbon Bala.lice The value of Qa- varied from 0,75 to 2,05 1ll moles/g./hr and the glucose concentration in the culture supernatant was never greater than 0. l%. Columns 6 ,7 and 8 of Table 3.2 indicate the proportional distribution of glucose carbon to cell substance, ethanol and associated co2 , and co2 via 52. non-ethanolic pathways such as the TCA cycle and the pentose phosphate pathway. Experiments performed in conjunction with those described here (Moss, Rickard, Beech and Bush, J.9691 have shown that c, the fraction of carbon in: dried cell mass of this organism is 0.48. Thus the fraction of glucose carbon fixed into cell substance (Table 3.2, column 6J is given by cµ 480µ y = = C 72 % -l Since µ did not vary from O.J. hr , except for states s1 and s 2 , there was an inverse relationship between Ye and QG• The fraction of glucose carbon which is directed to the products of ethanolic fermentation (Table 3.2, column 71 is 3~ or The fraction of glucose carbon directed to co2 by non-ethanolic pathways (Table 3. 2 , column 8) is given by the ratio The sum of the f'ractions in columns 6, 7 and 8 of Table 3,2 should give a value of J. if glucose is the sole carbon source and if the fate of glucose carbon has been completely accounted for in the fractions given. 53. The f'req_uent recording of' a sum greater than l (Table 3.2, column 9} suggests that some non-glucose carbon is being utilised. This could come f'rom the small amount of ethanol known to be in the medium (ea. 0.2% v/vl or f'rom the yeast extract and peptone components of the medium. Furthermore, the production of acid by the culture (Table 3,11 m~ be associated with excretion of' organic acid radicals. No measurement was made to indicate this avenue of carbon loss which, if accounted f'or, m~ have given even higher values f'or the carbon balance f'igure, Production of' Carbon Dioxide and Ethanol The value of QCO remained f'airly constant throughout the experiment, 2 In Table 3.2, column 3 shows the f'raction of the QCO value which was 2 associated with the production of' ethanol. This f'raction was greater than 0 only at the lowest values of' dissolved oxygen and its greatest value was 0,26. 3, 2 GRCYNTH IN STEADY STATE CONTINUOUS CULTURE AT VARIOUS LEVELS OF OXYGEN SO LurI ON RATE The response of C. utilis to growth at three progressively increasing o:xygen solution rates was studied. Under each set of' conditions, o:xygen solution rate was f'ixed as represented by the -values f'or gas f'low rates and stirrer speed. The -value of' dissolved oxygen was measured, but not controlled. -1 In the 3 1 culture, dilution rate was f'ixed at 0.1 hr , temperature was controlled at 30°c and pH at 5 .o. At each steady state JDeasurements were made of dry weight, cytochrome concentration, Qo2 , %o , ~, QG and the 2 rate of acid production. The data f'rom this study are shown in Table 3.3. In the three states which were studied, dry weight and Qo2 varied directly with mi:ygen solution rate, while QE and QG varied inversely with oxygen solution rate. At the highest solution rate studied (Table 3.3, state 3) ethanol was not produced, but fermentation of glucose to ethanol was evident at the lower oxygen solution rates. At the lowest oxygen solution rate (Table 3. 3, state l}, ~, QG and dissolved oxygen showed much higher values than in the other two steady states and the concentrations of the cytochrom.es and value of Q02 were very much lower, At this steady state, the organism appeared to have adapted to the conditions of low oxygen supply by developing ethanolic fermentation as a large component of its energy metabolism, 3.3 THE TRANSIENT STATE FOLLOWING A STEP CHANGE FROM HIGH TO LOW DISSOLVED OXYGEN 3,3.l The Nature of the Imposed Disturbance The behaviour of chemostat cultures of C. utilis was studied following th~sturbance of a steady state by the imposition of a step change in the dissolved oxygen value, Two experiments were performed, in each case the initial steady state being a 3 1 chemostat culture in which the dilution rate was fixed at O.l hr-1 , temperature was controlled at 30°c, pH at 5,0 and dissolved oxygen at 235 µM, In each of the experiments a different State Stirrer Gas Flow Dissol-ved Dry QG Qo Qco QE Cytochrome Speed (ml,/min.) Oxygen Weight 2 2 Concentration No. (r,p,m,) Q.lMI (g/11 (% of maximum) Air Oxygen Cm10.oles /g. ary wt/hr l A B C l 250 100 0 2,5 0,24 12.87 0,35 8.57 - .12,0 26.0 18.0 250 100 0 2,5 0.24 12,87 0,60 9.11 9.60 5,5 19,5 14.o 250 100 0 2,5 l,Ol 11,72 0,67 5.99 9,25 7,0 20,5 16,0 2 . 350 250 0 0,5 3.74 3,26 1.53 12,25 3,77 41.0 39 ,5 35.0 330 250 0 0,1 3.43 ],55 2,25 12,87 4,77 41.0 38,0 34,0 330 250 0 0,1 3,50 3.48 1,80 12.87 2.81 44,5 38,0 37.0 300 250 0 0,1 3.33 3,63 1,56 14.37 3.16 55.5 49.5 4o.o 3 600 450 50 0.1 10.5 0.81 3.23 2,21 0 600 450 50 0,3 12,5 O.85 2 .39 2.84 0 38,0 39 ,0 37,5 600 450 50 o,4 16.7 O.86 2,28 3,82 0 35,0 36,0 32.0 Table 3,3: Data from growth of C, utilis in steady state continuous culture, Oxygen solution rate was varied as indicated by stirrer speed and gas flow rates. Throughout, dilution rate was maintained at 0.1 hr-l, temperature at 30° and pH at 5, 0. Several readings of the parameters measured at each steady state are shown, V1 V1. 56, technique was employed to impose the disturbance, In the first, the set point of the dissolved o:xygen controller was adjusted from 235 µM to 1.2 µM, In the second, automatic control of dissolved o:xygen was discontinued and the oxygen solution rate was fixed at a low value. In both experiments, the result of the disturbance was a step change in dissolved o:xygen from a high to a low value, In each experiment, during the steady state prior to the change and subsequently until a new steady state had been reached, measurements were periodically made of the cytochrome concentration, dry weight, Q02 , QCO , 2 QG, ~ and the rate of acid production. 3.3,2 Transient State Following a Step Change in the Controlled Level of Dissolved 0:xygen In this experiment, the disturbance was imposed by altering the set point of the dissolved o:xygen controller from 235 µM to 1,2 µM. The readings for the various parameters are given in Tables 3.4 and 3.5 and Figures 3.4, 3.5, 3,6 and 3,7, The data for the initial steady state represents the latter stages of a steady state which had been maintained for several days, In this state, oxidative energy metabolism operates to the complete exclusion of fermentative pathways as evidenced by the absence of ethanol in the culture supernatant. 'lhe small amount of ethanol present in the sterile medium is being itself utilised. The para.meters of this steady state conform with the previous findings for the same growth conditions as shown in Table 3.l. Table 3,4 Time after Dissolved Dry Analysis of Culture QG Qo Qco QE Rate of Acid step change ()Jcy'gen Weight Supernatant 2 2 Production (hr) unl N Na0H/g, (µM) Ethanol fin (g/11 Glucose moles/g,dry wt/hr l dry wt/hr) (% w/v} (% v/v} - 35,5 235 13,80 0 0 0,59 - - -0,26 0,57 - 24.o 235 13,80 0 0 0.60 -- -0.26 0.60 - 17,5 235 13,31 0 0 0,62 -- -0,27 o.63 - 1.0 235 13.ll 0 0 0,63 18,88 3,85 -0,27 0,58 - 0.5 235 .13 ,28 0 0 0,62 23,59 2.10 -0,27 0.58 1,0 2,0 ll,82 0 0,04 0,69 - - -0,24 o.65 2,0 1.2 11.87 0 0,04 0,69 - - -0.24 o.63 3.0 l.2 12,53 0 0,05 o,66 - - -0.22 0.71 4.0 l.2 J..2,83 0 0 0,64 - - -0,28 0.69 5,0 1,2 13.00 0 0 0,63 - - -0,28 o.68 6.o 1.2 13,13 0 0 0,63 10.27 2.36 -0.27 0,56 7,0 l.2 12.56 0 0 0,65 10,71 2,47 -0,28 0.58 .. 8.o 1.2 13,12 0 0 0,63 -- -0.27 0,56 9.0 1,2 13,20 0 0 0.62 - - -0.27 0,56 10,0 l,2 12.94 0 0 0,63 - - -0,28 0,57 11.0 1.2 13,19 0 0 0,62 - - -0.27 0,56 12,25 1.2 13,31 0 0 0,62 l0,08 2,14 -0,27 0.55 13,0 l.2 ll,48 0 0 0,72 ll,75 2,65 -0,3l o.64 14,5 l,2 13.16 0 0 0.62 - - -0.27 0.71 Vl 25,0 l.2 8,77 0 0.24 0,94 15.82 6,35 0.08 0,59 -.:i. /Table 3,4 contd. Contd. ', -, '·, "' ·-, ·-...... '·-....._ ·,. ·,·, ·,, Dry Analysis Rate of Acid Time a:rter Dissolved of Culture QG Qo Qco ~ step change O:xygen Weight Supernatant 2 2 Production (ml N Na0H/g. (µM) (g/1} Glucose Ethanol Onmoles/g,dry (hr) wt/hrl. dry wt/hr) ($ w/vl (% v/vl 26.0 1.2 7 .24 0 0.81 1.03 - - 1.33 0.21 27,0 1.2 7 ,59 0 0,67 0,97 - - l.07 0,25 28,75 1.2 6.61 0 o.81 l.12 -- 1.60 0,28 30,0 1,2 6 ,03 0 0,62 l,22 3.00 28.73 1.21 0,31 31,0 l.2 5,32 0 o.4o l,39 - - o.65 0.35 32.0 l.2 5,07 0 0,89 l,46 - - 2.36 0,37 39 ,0 1.2 2!00 0,03 l,25 3!61 -- 9.09 0,87 56,0 1,2 1.85 0,05 - 3.84 -- - 0.90 l2l,75 1.2 3,o6 0 l,27 2,41 0.56 42,92 6.06 0,54 127.0 l.2 3,o6 0 l.18 2.41 3.13 32,62 5,55 0.61 151,0 1,2 3,37 0 l.23 2,19 3.15 25.00 5 .31 0.65 153.5 l.2 3,37 0 l.23 2.19 3,77 30,19 5 ,29 o.44 167,75 l.2 3,28 0 l,23 2,25 3.95 29.0l 5 .45 0.67 176,5 l.2 3.28 0 l,18 2,98 -- 5 .18 0.61 192.5 l,2 3,80 0 1.15 2.57 - - 4.34 0,54 Table 3 .4. Data :from growth of', C-. ut1..'1is. in continuous culture showing changes in metabolic activity following on imposed step change in the controlled -value of dissol-ved oxygen from 235 }JM to l,2 µM, Dilution rate 0.l hr-1, temperature 300c, pH 5.0. V1 -.;i .ID 58. Time * A:rter Cytochrome Concentration Step Change (percent of :maximum.I (hr) A B C -22.0 55.0 60.0 52.0 - 1.0 49 .o 56.0 50.0 1.0 42 .5 46 .o 42.5 2.0 40.0 45 ,o 40.0 3.5 39.0 44,5 41.0 5.0 44,o 50,0 45 .5 6.5 65.0 67,0 62.0 8,0 47,0 47,0 42.5 10,0 65.0 62.0 44.o .J.2. '0 56,0 62.0 50,0 13.5 44,o 42,5 40,0 25,0 62.5 67 ,5 57 ,5 27,0 62,5 65,0 57,5 29,0 91,5 95,0 83.0 31,0 60.0 62.0 54.0 53,5 20.0 23,0 23.0 122,0 40.0 37,0 33,0 125,0 41.0 43.0 35,0 148.5 60.0 57.0 50.0 151,0 60,0 50,0 50,0 1T-L,0 71.0 62,0 57.5 174.o 56,0 47,5 46.o 195,J 65,0 62,0 50.0 199,0 56,0 47,5 46.o 62,.5 218,5 " . 71.0 67.5 * Mid-point of collection period. Table 3.5, Changes in cytochrome concentration in C. utilis grown in continuous culture following an imposed step change in the controlled -value to µM. Dilution rate 0.l hr-1 , temperature of0 dissolved o:xygen from 2351,M 1,2 30, pH 5.0, , z 0 t-4 E--4 2 3 5 . Fig. 3, 4: Plots of (a) dis1olved oxygen and (b) dry weight against time after imposition of a step change in diaaolved oxygen from 235 pM to 1. ~pM. From data given in Ta~le l. 4. .. 0 50 Ooz • 9COz •• C 9o 40 J-4 ...... c= i 30 ~ 'tS • .....bO f •I) 20 -aa a ,; • ·10 I 0 1.9:::t:=:12:=:Q::=C::C~ca:ICC::::::::i:!~J....L.iJ:l:tla..::::J -3>-lQ. 1 · 10 100 • TIME AFTER STEP CHANGE (HR) Fia. 3. 5: Plots of 002, Ocoz and Oo again~t time after imposition of a itep change in dissolved oxygen from 235 pM ~o 1, 2 pM, From data given ln Table 3. 4. .• .I -', 10. Jot ...... d i 7. 5 t' 'tl s.o DO• ,. z. 5 ...•t» 0a s -a>-10 10 100 o. 50 o.zs 0 .a>-10 1 10 100 TIME AFTER STEP CHANGE (HR) _F_ia..,._3_.6_1 Plots of (a) OE and (b) acid production \ again1t time after impo1ition of a 1tep .. : '- change in di11olv~d oxygen from 235 pM to 1. 2 pM. From data 1lve11 in Table J.. 5. 100 CYTOCHROME A. 80 60 -~ ~ 40 ! ;_ 20 ~ '·' fz. . 0 0 1 10 100 '£,-4 -l/J-10 z . 100 (al CYTOCHROME B u ~ 80 (al ~ - 60 o•z ~ f-4 40 ~ f-4z 20 (al uz 0 0 -llJ .JO 1 10 100 u 100 CYTOCHROME C ~ 0~. 80 ::r: u 60 0 f-4 u>4 40 20 0 1 -a>-10 1 10 100 TIME AFTER STEP CHANGE (HR) ' Fig. 3, 71 Plots of concentrations of cytochrome• against time after impoaition of a atep change _in diaaolved oxygen from 235 pM ,. to l. 2 pM. From data 1lven ln Table 3, s. 59_, Changes in Dissolved Qxygen The high Qo2 at the time of imposition of the step change enabled the dissolved o:xygen value to fall rapidly i1!1lDediately the set point of the controller was changed. The new controlled level of 1.2 µM was attained in about 1.5 hr and was maintained for the remainder of the experiment. Changes in Respiratory Enzymes and 0ygen Uptake The cytochrome concentration in the cells remained constant until about 5 hours after the step change. From this point, fluctuations in the valueswere recorded until a new steady state was reached after 200 hr, Between 27 hr and 148 hr, the cytochrome values varied over the very wide range from 91.5 to 20.0. Following the disturbance, Q02 fell sharply from a value of approximately 20 m moles /g. /hr at zero time to approximately 10 m moles/g. /hr at 6 hr. Th.is was the first measurement of Qo2 after the disturbance and it is likely that the rate of fall was greater than that indicated in Figure 3,5. After the initial drop in Qo2 , variation in the values recorded follow variations in cytochrome concentration, At the new steady state the value of Q02 was 3,95 m moles/g. /hr, very much lower than in the initial steady state, Appearance of Fermentati ve Metabolism Fermentation of glucose to ethanol and carbon dioxide was not evident until 20 hotll's after the step change, when the rates of output of the products 60. of fermentation began to rise. These rates reached maxima at between 40 and 80 hr and then fell awey to their values at the new steady state, in which fermentation was a significant component of energy metabolism. changes in Dry Weight and Glucose Uptake Dry weight remained constant for 10 hr after the disturbance and then suffered a sharp fall as the culture began to wash out, The new steady state value for dry weight was very much lower than in the initial steady state. The fall in dry weight is associated with the development of fermentative metabolism and resulted in an increased value for QG. The value for the concentration of glucose in the culture supernatant was virtual]y zero (less than 0.01%) throughout the experiment. Evidence for Active Breakdown of Cytochrome In this transient study, during the 24 hr period from 29. 0 hr to 53,5 hr after the imposition of the disturbance, the value of cytochrome A fell from 91, 5 to 20.0. Cytochromes B and C showed a similar change. From a knowledge of the dilution rate and the changes in the dry weight of the culture over this period it can be shown that the fall in the measured value of cytochrome A was part]y the result of active breakdown of cytochrome by the cell. For the cytochrome A value to fall, the rate of biosynthesis of cytochrome A must be at least reduced, Let us assume the extreme case where cytochrome biosynthesis ceases altogether after the point where a cytochrome A value of 91.5 is recorded, the cells formed after this point having a 6J., cytochrome A value of 0. Let us further assume that the mean dry weight of a cell remains constant during the subsequent changes , and that the culture represents a perfect model of a continuous stirred reactor, Now, regardless of the value of the specific growth rate, the rate of loss from the culture of the high cytochrome cells will follow the sin:qile washout kinetics given by dx = - Dt dt i.e. = x e-Dt 0 If at time zero the whole of the mass of the culture consists of cells with. a cytochrome A value of 91,5, then at any interval a~er time zero the contribution of the high cytochrome cells to the dry weight value of the culture can be calculated, The ratio of this contribution to the remaining dry weight value is the ratio of high cytochrome cells (A= 91,51 to zero cytochrome cells, Thus the cytochrome concentration can be predicted, Figure 3, 8 shows the changes in cytochrome A and dry weight values during the period of fall in cytochrome A from 91.5 to 20,0. Although. only three experimental values were available, the fall in cytochrome A followed a smooth curve. The changes in dry weight however did not follow a smooth curve and the more frequently measured turbidometer readings were used to indicate the shape of the curve. The plots in Figure 3.8 were used to provide a series of values for cytochrome A and dry weight for the 24 hour Fig. 3. 8: Plots of cytochrome A concentration, dry weight and turbidity from a section of the data from Tables 3. 4 and 3. 5 between 29 and 53 hra. The more frequently mea1ured turbidity value• were used to indicate the shape of the dry weight curve. CYTOCHROME A CONCENTRATION (percent of arbitrary maximum) 0 r 0 0 0 -0 co N . ,. \.. ' I .. :· ~- ' J i ! ' ; .. ~. 'f • . . .. ' ''"" o ~ a· (t/8) J.H0l3h\ Alla 0 N. . . 00. 0 .. 0 0 ,' 0 0 .- ()NJQV:!1f ll::1.t3"0cmlll n.t 62, period under consideration. Table 3.6 shows these values and also the predicted values calculated on the basis of washout. A sample of this calculation is given in Appendix II. The experimental dry weight values in Table 3.6 are always higher than the predicted values, indicating that 1.1 has a positive value. The cytochrome values however are consistently lower than the values predicted on the basis of washout of the high cytochrome cells. This indicates that the cytochrome A value of 91,5 in the original cells is not being maintained and that active breakdown of cytochrome by the cells is occurring. Further, it is likely that the cells formed after time zero would have some cytochrome and not a value of 0, as assumed. In this case the predicted cytochrome A value would be even higher, and active breakdown of cytochrome would be contributing to a gr-eater extent to the overall fall in cytochrome A which is observed. In addition to predicting overall change from time zero, Table 3.6 also shows the predicted values of cytochrome A calculated on the basis of the dry weight and cytochrome A value at the beginning of each interval of measurement, The differences between these figures and the measured values of cytochrome A show that during the first 10 hours active cytochrome breakdown is contributing to the fall in cytochrome A value to a greater extent than during the period f'rom 10 to 20 hours, where the predicted value approximates the measured value. Time a:rter Dry Weight (g/11 Cytochrome A Concentration c o:mmencement (percent of arbitrary maximuml of fall of Value predicted Value predicted cytochrome A Measured from washout (hr) Measured from washout Value Value From time From previous From time From previous zero measurement zero measurement 0 7.59 7.59 ... 91.5 91.5 l.75 6.61 6,34 6,34 10.0 87 .5 87,5 3,0 6,03 5,62 5.80 60.0 85,3 67,5 5,0 5,07 4.60 4,93 48,o 83.0 58,5 10,0 3,40 2,78 3,08 31,5 74,7 43,0 20,0 l,90 l,02 l,25 22,0 48.0 20,0 Table 3.6: Section of data from Tables 3,4 and 3,5 showing changes in cytochrome A concentration and dry weight between 29 and 49 hours after imposition of the step change, Measured values which do not appear in Tables 3.4 and 3,5 have been' obtained graphically from Figure 3,8, 0\ w. 64. This treatment of the data is not invalidated by the notion that during the budding cycle, the mother cell 1nay transfer some of its cytochrome complement to the daughter cell, thus effectively reducing the cytochrome concentration in the original cells. The argument remains valid because, regardless of the distribution of cytochrome between mother cells and daughter cells, the total a.mount of cytochrome per unit of cell 1nass will be constant at a given time, and this is the figure being considered. 3.3.3 Transient State Following a Step Change from a Controlled Dissolved 0ygen Value of 235 µM to a Fixed Low 0ygen Solution Rate To effect the step change in this experiment,automatic control of dissolved oxygen was discontinued and a fixed low oxygen solution rate was immediately substituted, This oxygen solution rate was represented by a stirrer speed of 600 r,p .m. and an air flow of 250 ml/min. The readings for the various para.meters of the culture in the initial steady state and during the transient period until a new steady state was reached are given in Tables 3.7 and 3.8 and Figures 3.9, 3.10, 3.ll and 3.12. In the initial steady state, where dissolved oxygen was controlled at 235 µM the culture again showed a similar pattern of metabolism to previous findings under these conditions. Changes in Dissolved 0ygen In this experiment the value of the dissolved o.xygen was uncontrolled following the disturbance. However, as in the first transient experiment, Rate acid Time after Dissolved Dry Analysis of Culture QG Qo Qco ~ of step change Oxygen Weight Supernatant 2 2 production (µM) (ml N NaOH/g. (hr} (g/1) Glucose Ethanol m moles/g. dry wt/hr dry wt/hr) (% w/vl (% v/vl -15,0 235 10,ll 0 0 o.88 17 .50 2.51 -0.35 0.75 - l.O 235 10.14 0 0 o.88 18.10 2 .93 -0,35 o. 74 8.0 o.8 7.61 0 0,50 l,35 1,70 2,34 0,69 0,74 9,5 o.8 6,64 0 0,52 1.55 1,66 3,71 0,84 0,50 10,5 1,0 6,41 0 O, 52- 1,60 1,74 3,47 1,06 0,52 ll,75 1,0 5,96 0 0.62 l,72 1:84 3,78 1,24 0.56 13,5 1.0 5,73 0 0,60 l,79 l,94 4,20 l,22 0,50 14.5 1.0 5,72 0 0.60 l,79 1,96 4.12 1.20 0,50 16,5 1.0 5,64 0 0,63 l,82 l,99 4,18 1,33 0.60 18,0 0,9 5,69 0 0,62 1.80 l,93 3,96 1,29 0,59 20,0 0,9 5,77 0 0.61 l,78 2,19 3,97 1.25 o.64 33.0 0,9 5.24 0 0,71 1.96 2.22 4,09 l,70 o.66 34,5 l.1 5,00 0 o.66 2,05 2,26 4.29 1,59 0.80 36,5 1.1 5,00 0 o.66 2,05 2.28 4,19 1,59 0.80 38,0 1.3 5,22 0 0,60 1.96 2,25 4,05 1,36 o.64 39 ,5 1.3 5 ,3l 0 0,62 1,93 2.22 3 ,94 1,37 0.63 56,5 0.9 5, 46 0 0,67 1.88 2.29 4.24 1,59 o.65 59,0 1,0 5,41 0 o.64 1.89 2.10 4.34 1,42 0.62 63,0 1.0 4,99 0 0.72 2,05 2,54 4,79 1.83 0,67 83.5 1.0 6,75 0 0,36 1,52 2,05 3,72 1,42 o.42 86,5 1.0 6,28 0 o.46 1,63 2.10 4.13 1.73 0,67 Table 3,7: Data from growth of C, Utilis in continuous culture showing changes in metabolic activity following an imposed step change from a controlled dissolved o:xygen value of 235 µM to a fixed o:xygen solution rate represented by an air flow of 250 ml/min and a stirrer speed of 600 r,p,m, Dilution rate 0,1 hr-1, temperature 30°, pH 5,0, 0\ VI 66, Time * after Cytochrome Concentration step change (Percent of a.rbi tra.ry maximum} (hr1 A B C -13,5 32,5 47 .o 37.5 - l.O 37.0 47.5 40.0 8,5 37,5 52.5 41.0 9,5 38.5 47,0 42.5 .11. 0 41. 0 57.0 45.5 .13,0 42,5 52.5 44,5 14.5 51.5 67,5 52.0 16.0 54.o 70.0 55,0 18,0 57,5 56,5 46.0 20,0 68.o 79,0 62.5 34.o 56.0 59 ,0 42.5 36.5 70.0 65,0 47,5 38.5 65.0 65,0 45.0 57.0 73.5 65.0 45.0 60.0 73.5 62,0 45 .o 82,5 81.0 67,0 47 .o * Mid-point of collection period, Table 3,8: Changes in cytochrome concentration in C. utilis grown in continuous culture following an imposed step change from a controlled dissolved o:xygen value of 235 µM to a fixed o:xygen solution rate represented by an air flow of 250 ml/min and a stirrer speed of 600 r .p ,m, Dilution rate 0.1 hr-1 , temperature 30°, pH 5,0, 235M <•) I I I I I . I I I 4 l 3 • .. .. . 2 1 0 .__..__ ___._ ...... ,,,...... ,,...... _..,_...._..L...... L.L..U -a> -10 1 10 '100 • '. • I ..• 2 0 .a>-10------....&,--&.11,,--- 1 10 ...... ~100.... ' TIME AFTER STEP CHANGE (HR) Fl1. 3. 9: -Plot1 of (a) dh1olved oxygen and (b) dry I iweight again1t time after impo1ition of ' a 1tep change from' a controlled di11olvecl : !' oxygen value of a35 pM to a f~ed low oxy1en ·,olutlon ,rate. Ftom cla~ 1lven in Table 3, 1, · 20 .....] 15 i .,,t- • IQ ',' .....tlO •u "o . a s 0------...... ----....______~-10 1 10 100 TIME AFTER STEP CHANGE (HR) Fig, J, 10: Plota of 0 02, Ocoz and Oo again•t time after imposition of a atep change from a ; ·. Controlled di••olved oxygen value of 235 pM . ' ' . to a fixed low oxygen •olutlon rate. From . .t&ta aiven la Table 3, 1 • r' 2.0 (a) ,...... d 1. 5 i t' 1.0 ~ • ...... tl8 o.s ....•u 0 0 8 8 -o.s -a>-10 l 10 100 1. 00 (b) o.7s o. so 0.2s • 0 ...... _...... _ ___,__._....,_....,_...._ _ _.___,___._.....,,_, ...... a>-10 1 10 100 • ~ TIME AFTER STEP CHANGE (HR) Plots of (a) OE a~d (b) acid production against time after imposition of a •.tep change from a controlled di11olved oxygen value of 235 p.M to a fixed low oxygen 1olution rate, rrom data aiven ta Table J. I, 100 CYTOCHROME A 80 60 40 20 0 .....-.11.___..,_.a...,.i ...... L.LL._...... &.I~ -al•IO 1 10 100 100 CYTOCHROME B 80 60 40 20 0 '--1....ll'---.._,._...._~1,1,1.,-...... a, -10 1 10 •• 100 . 100 ,, CYTOCHR.9ME C ,.. 80 • zo 0 ..... __..___.,___._...... ~----- ...... &.1,1 .a,.10 l 10 100 TIME AFTER STEP CHANGE (HR) Fla. i.121 Plota of concentratlonl of cytochrome• againat time after lmpoaition of a 1tep change from a controlled dilaolved oxygen value of 235 ,aM to a fixed low I oxygen •~lu~lon rate, ~r~m data 1lveb I I ,._ ...... ;.., .. 1. Ill 67. th.e dissolved oxygen fell rapidly and a value of approximately 1.2 µM was reached in l hr. This value remained steady until about 15 hr. After this time some small variations occurred but the dissolved oxygen value did not change significantly for the remainder of the experiment. Changes in Respiratory Enzymes and Oxygen Uptake .As in the first transient experiment the cytochrome concentration remained unchanged for some hours before exhibiting fluctuations which finally led to the,hew steady state level. The amplitude of the fluctuations in the cytochrome value was much less than in the first transient experiment and the new steady state was attained by 100 hrs, half the time required for a new steady state to be reached in the first experiment. The Q02 value had fallen to less than 2 m moles/g./hr within 8 hours and did not rise for the remainder of the experiment. This was a much more rapid fall than in the first transient experiment. Appearance of Fermentati ve Metabolism Production of ethanol was evident at 8 hr in this experiment, much earlier than in the first transient experiment. ~ increased steadily from this time until the new steady state value of 1.73 m moles/g./hr was reached at 90 hr. This value was low COlllpared with the new steady state QE value of 4,34 m moles/g,/hr in the first transient experiment • .Associated with the increase in QE was a rise in QC02 which rose to a maximum value of 4.79 m moles/g./hr,only about one tenth. of the maximum QC02 recorded in the 68, first transient experiment, The new steaay state. value for QCO was 2 4.13 m moles/g./hr compared with a value of 29.0l for the first experiment, Changes in Dry Weight and Glucose Uptake In similar fashion to the other metabolic parameters, dry weight showed the same pattern of change seen in the first transient experiment, but the changes began earlier, their amplitude was much smaller, and the new steaay state value was reached much earlier. The lowest dry weight value recorded was 4.99 g/1 compared with a minimum value of 1.85 g/1 in the first experiment. Changes in the value of QG corresponded closely with the changes in the dry weight, but inversely. As the concentration of glucose in the culture was virtually zero (less than 0,1% w/v} throughout the experiment, the fall in dry weight and increasein QG recorded represented a fall in the yield of cell material with respect to glucose, and this was associated with the development of fermentative energy metabolism. 3.4 EFFECTS OF OZONE ON C. UTILIS 3,4.l Effect of Ozone on the Cytochromes of a Resting Cell Suspension Cells from a chemostat culture of C. utilis which had been grown at a dilution rate of 0.1 hr-l and with temperature controlled at 30°c and pH at 5. 0 were washed once and suspended in M/15 phosphate buffer at pH 7 .o. A volume of 2 1 of the suspension was placed into an open vessel consisting of a large conical glass filter funnel with the upper end of the outlet tube closed by a rubber bung. A glass tube passing up the outlet tube and through the rubber bung enabled passage of gas through the suspension. A laboratory stirrer was fitted to the vessel. A stream of mcy-gen containing 1000 p.p.m. (0.1%) of ozone was passed at a rate of 300 ml/min through the suspension at room temperature and the cytochrome concentration in the cells was measured at intervals. Table 3.9 shows the results of this experiment. Ai'ter a slight initial rise, the concentration of cytochromes had reached 0 a:rter 18 hr. Time of Cytochrome Concentration Exposure (percent of maximum} (hrJ A B C 0 37,0 30.0 29.0 3 46,o 41.0 38,5 6 47.5 42 .5 37,0 18 0 0 0 Table 3,9: Changes in cytochrome concentration in c. utilis with time of exposure of 2 1 of a washed cell suspension to 300 ml/min of oxygen containing 1000 p.p.m. of ozone. Another washed suspension of cells (from a chemostat culture of C. utilis grown as above and washed and suspended in phosphate buffer} was divided into three volumes each of l 1. Each was placed into an open vessel as described in the above experiment. The first was treated with the passage of 300 ml/min of oxygen containing .1000 p .p .m. of ozone. The 70. other two volumes were used as controls and treated with (a) passage of 300 ml/min of OJcygen without ozone and (b} stirring only without passage of gas. Samples were taken periodically from all three vessels and the concentration of cytochromes in the cells determined. Table 3.10 shows the results of this study. 'Ihe cytochrome concentration in the cells of the control samples remained unchanged over a period of 22 hours. 'Ihe ozone treated cells however showed an increase in cytochrome concentration up to 6 hr followed by complete loss of cytochromes a:rter 22 hours. 3.4.2 Effects of Ozone on Cytochromes and Viability of a Non-Growing Culture C. utilis was gr-own in chemostat culture at a dilution rate of 0.1 hr ~ and with the temperature controlled at 30 0 C and the pH at 5 .o. The dissolved oxygen value was approximately 50 µM and the steadyt:ltate dry weight value was 12. 07 g/1. When a steady state had been established, 1.5 1 of the culture was taken and placed in an open vessel of the type described in section 3.4.1 at room temperature. Since the steady state glucose concentration in the culture supernatant was virtually O, gi:-owth of the cells in the sample could not occur a:rter its removal from the culture vessel. The sample of whole culture was subjected to passage of l.5 1/min of o:xygen containing 170 p.p.m. (0.017%} of ozone. This represents a flow rate of 0.25 ml/min of ozone. The concentrations of cytochromes in the cells and the percentage viability were measured periodically. Time of Cytochrome Concentration Exposure (percent of maxilll1.llt1} (hr} Standing ()Jcy'gen 0Jcy"gen (300 ml/min) (300 ml/min} containing ozone (1000 p.p.m.} A B C A B C A B C 0 38.5 38.5 35.0 38.5 38,5 35.0 38.5 38,5 35,0 4 37.0 39 .5 36,0 41.0 34.5 34,o 44.5 44.5 41.0 6 38.5 42.5 36,0 37 ,0 31,0 33.0 70.5 67 .o 53, 5 10 77.5 46.o 41.0 35,0 30,0 34.o 67 .5 33,0 36.o 22 42.5 50.0 46.o 32,5 30,0 30,5 0 0 0 Table 3,10: Changes in cytochrome concentration in a washed suspension of C.utilis on exposure to ozone compared with exposure to OJcy"gen only and with a standing suspension. Volume of each suspension, .l 1. 'p. 72, The results of this study are shown in Table 3,ll. Cytochrome concentration began to decline on passage of ozone. Although slight increases in the concentrations of cytochromes Band C were recorded at l hr, these were not as marked as th.e general increase in cytochrome concentration found in the ear]¥ stages of ozone treatment of a washed cell suspension (section 3. 4.l). The concentration of all cytochromes gradual]¥ fell to O and a definite order of their disappearance was established this being C, followed by A with B the last to disappear complete]¥. Cytochrome C had dis appeared at 3. 75 hr and after 7 hr all cytochromes had completely disappeared. The results reveal a rapid loss of viability when cells are treated with ozone at this level. 3. 4.3 Effect of Ozone on Cytochromes and Growth Rate of a Turbidostat Culture A turbidostat culture of C. utilis was set up. The culture volume was 3 1 and the temperature was controlled at 30°c. pH was measured but not controlled. The turbidity controller was set to control at a dry weight value of approximately 4 g/1. 0Jzygen was passed into the culture at 120 lJl.J./Illin, the stirrer speed being set at 300 r.p.m. Itwas not possible to measure the dissolved o::xygen value in the presence of ozone as earlier attempts to do this had Led to rapid deterioration of the o:xygen electrode and disintegration of its pozyth.ene membrane. When a steady state h.ad been attained the ozone generator, through 73. Time of Cytochrome Concentration Viability Exposure (percent of arbitrary maximum} (percentl (hr) A B C 0 41..o 41..o 37.0 99 l.O 41,0 56,5 39.5 0 2,5 27 .5 24.5 l8.0 0 3.75 l2.0 15.0 0 0 5,0 0 trace 0 0 6.o 0 trace 0 0 7,0 0 0 0 0 Table 3.ll: Changes in cytochrome concentration and percentage viability in a sample of whole culture of ·c. utilis exposed to ozone. Volume, l.5 l; cell dry weight, l2 ,07 g/1; flow rate of gas, l .5 1/min of oxygen containing l70 p.p,m, of ozone. which the oxygen stream passed, was switched on and the variable shield adjusted to a slight]y open setting. The responses of the cytochrome concentration and the growth rate were measured, and the ozone dosage was gradual]y increased by adjusting the ozone generator or the oxygen flow rate. This was continued 1IDtil growth ceased, Table 3 .l2 shows the results of this experiment. At the lower levels of ozone treatment, cytochrome concentration showed a slight increase as was folIDd in the,4,ashed cell suspensions (section 3.4.l). With increasing ozone dosage however cytochrome concentration decreased 1IDtil the cytochromes eventually disappeared altogether, Growth rate decreased in response to ozone +="" +="" . . --1 --1 utilis utilis 75 75 C. C. pH pH 3.8 3.8 4,7 4,7 3,15 3,15 2.8 2.8 2.6 2.6 2.9 2.9 2. 2. 2,8 2,8 3,l5 3,l5 of of .J.2 .J.2 0 0 - - - µ µ 0,05 0,05 0.016 0.016 0,07 0,07 0,l3 0,l3 0.l0 0.l0 0 0 0.002 0.002 (hr-ll (hr-ll culture culture ,5 ,5 C C - 0 0 - 77,5 77,5 62.3 62.3 69,0 69,0 69,0 69,0 97 97 100,0 100,0 l00,O l00,O 100.0 100.0 maximum) maximum) turbidostat turbidostat a a of of Concentration Concentration pH pH .5 .5 - 0 0 - B B arbitrary arbitrary 54 54 85,5 85,5 67,0 67,0 93,5 93,5 65,0 65,0 62.5 62.5 95,0 95,0 and and l00.0 l00.0 of of rate rate Cytochrome Cytochrome 5 5 (;percent (;percent A A 0 0 - - 89. 89. 80,5 80,5 29,0 29,0 77,5 77,5 growth growth 56,0 56,0 54,o 54,o 60,0 60,0 60.0 60.0 in in concentration, concentration, 0 0 0 0 - - 0 0 - - - ozone ozone 540 540 560 560 6l0 6l0 (p.p.m.l (p.p.m.l o:xygen o:xygen of of Concentration Concentration cytochrome cytochrome flow flow in in l20 l20 rate rate J.20 J.20 150 150 150 150 J.20 J.20 150 150 l20 l20 l20 l20 l50 l50 l50 l50 .l20 .l20 ozone. ozone. (ml/minl (ml/minl 0Jcygen 0Jcygen Changes Changes to to 3.12: 3.12: of of 5 5 0 0 exposure exposure (hr) (hr) 44 44 40 40 3l 3l 28 28 21 21 l0 l0 15 15 - 3 -27 -27 exposure exposure on on Time Time Table Table 75, treatment, growth having ceased altogether when the cytochromes had disappeared. The value of pH, initially 2 .8, showed an increase to 4. 7 as growth rate decreased. 3,4.4 Effects of Ozone on Cell Density, Viability. Cytochroni.es, Metabolism and Cell Fine Structure of a Chemostat Culture A chemostat culture of C. utilis was set up. The culture -1 volume was 3 1 and the dilution rate was fixed at 0.1 hr • The tempera- ture was controlled at 30°c and the pH at 5 ,0, Dissolved o:xygen was not measured because of the deterioration of the electrode in the presence of ozone. A mixture of o:xygen and air was passed into the culture and the stirrer speed was set at 600 r ,p ,m. The o:xygen stream was passed through the ozone generator, not operating at this stage, and mixed with the air stream before entering the culture vessel. Air was nrl.xed with the ozonised o:xygen stream since the method of calculation of Q and QCO requires that 0 2 . 2 the gas stream should include a non-reactive component, this being nitrogen. Measurements were made periodically of the dry weight, viability, cytochrome concentration, Q0 , QG and QCO • Ethanol was not detected in the culture 2 2 supernatant at any stage of the exper inent. A:rter a steady state had been established in the absence of ozone, the ozone generator was switched on and adjusted to give a low output of ozone. A:rter measurement of the para.meters of the steady state reached under these conditions, the ozone input to the culture was gradually 76, increased by adjusting the ozone generator or the gas flow, At each level of ozone treatment the parameters of the steady state were measured over a period of several days and this procedUTe was continued until, at a high level of ozone treatment, the culture began to wash out. At each of the states studied, samples of the culture were taken for examination of thin sections of the cells by electron microscopy. The results of this study are shown in Table 3,13 and Figures 3,13 and 3.14. This actively growing culture showed a much greater resistance to ozone than the non-growing culture described in section 3, 4.2, where a flow rate of 0,25 ml/min of ozone led to complete loss of viability a~er 1 hour. On this basis, the chemostat culture described here, in which the mean residerretim.e of the cells is 10 hours , would be expected to show some effect from a considerably lower level of ozone treatment. This was not the case however and there was no significant effect of ozone below the level used in the non-growing culture, that is for states 1 to 6 (Table 3.131. At state 7, where ozone was being admitted to the culture at 0,38 ml/min., dry weight and Q02 had increased appreciably, but a fall in the concentration of cytochromes and in the value of QG had occurred. At state 8, where the ozone input had been increased to 0.48 ml/ min, the values of dry weight and Q~ showed further increases but the percentage viability, within the limits of accuracy of the method, had fallen only slightly from the value obtained before pass age of ozone. In state 9, the OJcy"gen flow rate through the ozone generator had been increased in an State Gas Flow Ozone in Dry V.iabili ty QG Qo Qco Cytochrome No. (ml/min} inflowing Weight (percentl 2 2 concentration gas (g/1} Gn1lloles / g • dry wt /hr J (percent of ' ' arbitrary maximum) Air Oxygen wn,. ml/min, A B C l 450 50 0 0 10,24 92,3 0,92 2.86 2.78 28,5 32,0 31,5 2 450 50 n4 0.06 10,71 96,l 0,89 2 .49 2,70 33,5 37,0 33,5 3 450 50 90 0.05 l0,19 95,7 0,80 3.10 2 ,90 28,5 36,0 33,0 4 450 50 l20 o.o6 l0,40 91,0 0,82 2 .36 2,96 27,0 34,5 30,5 5 450 100 208 0,10 9,79 90,6 o.85 4,36 3,17 30,0 34.o 32 .5 6 280 150 363 Q,16 13.25 75,4 0,67 3,50 3,96 27,5 34.o 28,5 7 145 335 700 0,38 17,73 74,8 o.48 7,14 1,89 16,5 23,0 21.5 8 100 540 750 o.48 20. 75 82.2 o.41 14,36 o.68 16.0 25.5 22.0 9 100 700 542 0,43 Time of 16 12.89 53,5 o.64 - - 16.0 28.0 26.0 exposure 44 4.63 5,5 l,43 76,03 o. 79 0,5 17,8 6.o in 51 4,02 4.o 1.29 84.90 0,75 0,2 6,5 0.2 state 9 (hr} 64 2,96 0,8 -- - 0 0 0 Table 3,13: Data from steady states of a chemostat culture of C, utilis exposed to gradually increasing levels of ozone, Dilution rate 0,1 hr-1; temperature, 30°; pH 5,0, Values shown are averages of several readings at each steady state except for state 9, where progressive changes with time are shown. ....:i ---.:J. 78, effort to f'urther increase the rate of ozone admission to the culture, but the upper limit of the ozone output of the system had been reached, However, although the rate of flow of ozone in state 9 was approximately the same as for state 8, a steady state was not reached due to a decline in the percentage viability and washout of the culture, During washout, in state 9, the concentration of glucose in the culture vessel, previously less than 0,::1%, rose to values of O. 46% at 44 hours and O. 72% at 51 hours. Cytochrome concentration fell progressively to zero during the washout period. It is likely that the value of dissolved oxygen was higher after the alteration of the oJcy"gen flow rate at the beginning of state 9, and this suggests greater sensitivity of C. utilis to ozone with increasing levels of dissolved oJcy"gen. During the washout period in state 9, exceptionally high values of Qo2 were recorded, These readings of Qo2 were based on the total dry weight without regard to the percentage viability, and considering the very low viability (less than 10%) and cytochrome values (0.2 for cytochrome A), can probably be regarded as largely representing combination of o::x;vgen somehow other than as part of a metabolic :function of the cells. Except for the latter stages of state 9, ozone was not detected in the effluent gas stream in the chemostat experiment. Ji':t~'-3.TI! Electron micrographs- of cells of C. utilis grown in < . continuous c1,1lt-ure without passage of ozone, from s-tate l of the experilnent described in section 3,. 4, 4 and Table 3.13. M = mitochondrion, Magnifications--! (al and (b I, x 45 1 000; (c 1, :x 40 ,ooo, F i g . 3 . 13 (a). Fig . 3. 13 (b ). Fig. 3. 13 (c ). Fig. 3.14: Electron micrographs of ozonised cells of C. utilis from (al state 8 1 (b} state 9, 16 hr and (c) state 9, 44 hr of the experiment described in -section 3,4,4 and Table 3,J..3. M = mitochoncrrion, Magnifications: (al, x 35 ,000; (b l, x 45 1 000; (cl. x 30 ,ooo, F i g. 3. 14 (a). • Fig. 3. 14 {b). Fig. 3. 14 (c) . 79. Effect of Ozone on Cell Fine Structure Figures 3,13 and 3.14 show electron micrographs of thin se~tions of cells of c. utilis from normal and ozonised states of the above experiment and are representative of many samples studied. In state l, before introduction of ozone to the culture (Fig. 3.13), the cells were seen to contain numerous well developed mitochondria. In states 2 to 8 of Table 3.13, the general appearance of the cells and their mitochondria remained unchanged from those shown in Fig. 3.13. In state 8 (Fig. 3.l4(a)} however, and subsequently the internal structure of the cells showed an i.meven appearance, but in state 8 :mitochondrial menibranes could still be discerned. In state 9 (Fig, 3.14 (b} and (c)l there was a progressive disruption of the internal structure of the cells i.mtil at 44 hr (Fig, 3.14 (c}l there was no evidence of mitochondrial structure. Because of the low percentage viability at this point, most of the cells examined would have been killed by ozone. Although the interior of the cells presented an amorphous appearance, the cell wall had not been ruptured by ozone. Ozone as a Mutagenic Agent Throughout this experiment in chemostat culture, the samples which were routinely plated onto nutrient agar and malt agar for detection of conta:minants were also plated onto Nagai 's medi1.llll (Nagai, 1965). This medium contains sodium tellurite and has been useful for the isolation of respiratory deficient mutants in cultures of S, cerevisiae. When grown on Bo. this medi1.llll, normal colonies are black due to reduction of the telluri te, while mutant colonies appear colourless. 'Ihe results from this provided no evidence that ozone acts mutagenic ally to produce respiratory deficient mutants of C. utilis. 8J.. 4, DISCUSSION · AND CONCIUSIONS 4.1 COMPARISON OF STEADY STATE RESPONSE OF C. UTILIS TO DISSOLVE) OXYGEN VALUE AND OXYGEN SOLUTION RATE Over most of the wide range of variation in o:xygen environment in which C. utilis has been grown, there was no relationship between the oxygen conditions and the level of respiratory activity. It was at the extremes of the range however, where oxygen supply was very low or where the value of dissolved oxygen was very high, that the organism responded to changes in oxygen conditions. The results from steady states with controlled values of dissolved oxygen (Table 3.11 indicate that a dependency of Qo2 on dissolved oxygen occurs only at very low levels of dissolved oxygen, these levels being at the limit of measurement with the Mack.ereth electrode, or at extremely high values of dissolved oxygen. Johnson (1967) grew C. utilis in chemostat and found a dependency of Q02 on dissolved oxygen up to a value of 2 .5 1-1M. However in Johnson 1s experiments the value of dissolved oxygen was not controlled but was varied by varying the dilution rate and therefore comparison with the results described here is of limited value. Harrison and Pirt (1967) found that in chemostat cultures of Klebsiella aerogen:es maximum oxygen uptake occurs at low OJcy"gen tens ions. However in our experiments with C. utilis, the increased cytochrome levels at low values of dissolved OJcy"gen were not accompanied by an increase in Qo2 • The maximum 82. Qo2 value was in fact recorded at a yery high value of dissolved oxygen. The experiments with varying oxygen solution rates (Table 3.31 produced a significant finding with regard to the relation between the dissolved oxygen value and the level of respiratory activity. This is that when the culture had adapted to conditions of -very low oxygen solution rate, a large proportion of the energy requirement is provided by fermenta ti ve break.down of glucose so that the oxygen requiremerlfor respiratory activity is very low. This can result in a rise in the dissolved mcy-gen value (Table 3.3, state .l) but under such conditions this relatively high level (2. 5 µM) of dissolved OJo/gen does not influence the cell to revert to the exclusively oxidative state which was observed when dissolved oxygen was controlled at values above 1.5 µM (Table 3.1). The fundamental difference here is that under conditions where the value of dissolved oxygen is controlled, the cell's oxygen requirements, however large, will always be satisfied, whereas at a very low oxygen solution rate the rate at which the cells may take up oxygen is lilllited and if all of the available energy source is to be utilised, the cell must adopt fermentation as part of its energy :metabolism. It is a characteristic of c. utilis in a glucose-limited chemostat culture that only at very low conditions of oxygen supply does fermentation appear at all, By comparison, Sacchar01gyces carlsbergensis, which has been studied in ch.emostat conditions silllilar to those described here for C. uti lis (Moss, Rickard, Bush and Caiger, .1969) displays fermentation of 83. glucose even at a dissolved oxygen value of 235 µM. However when c. utilis is grown under steady state conditions where glucose is not the limiting nutrient, but is maintained at a concentration of 100 to 300 mM (Moss, Rickard, Beech and Bush, 1969 l, fermentation occurs at all levels of dissolved o.xygen up to 235 µM. In these high. glucose conditions however, cytochrome synthesis has been shown to be limited by catabolite repression, which further indicates that fermentative pathways operate in C. utilis only when there is some limitation on the rate at which oxidative metabolism may proceed. Through all the steady states studied, the cytochrome concentration did not show any consistent relationship with either Qo2 or dissolved o.xygen. At the lowest levels of controlled dissolved o.xygen, the concen trations of the cytochromes showed a 50% to 75% increase while Qo2 did not change significantly, At the lowest o:xygen supply rate studied (Table 3, 3, state l}, Q02 and cytochrome concentration were very low compared to their values in other steady states, while dissolved oxygen showed a value of 2.5 µM. This latter situation simply represents the minor role of oxidation in the overall energy metabolism of the cell under these conditions. However it seems that the steady state at a dissolved oxygen of 0.15 µM and those at sj_ and s 2 (Table 3,11 represent states where energy metabolism is almost completely oxidative only if the cell possesses relatively large amounts of its respiratory enzymes. The steady state s1 , where dissolved 84, oxygen is below the limit of measurement, ... withthe Mackereth electrode appears to represent an optimum oxygen condition for cytochrome synthesis. This finding agrees with several earlier reports of the occurrence of maximum cytochrome levels under conditions of low oxygen availability (Moss 1952, 1956; Lenhoff, Nicholas and Kaplan, 1956) . Wimpenny (1967 l found that in chemostat cultures of E. coli, maximum levels of cytochro:me,<~1... could be obtained using nitrate or nitrite or oxygen as the terminal electron acceptor but at an approximately constant ~ value. These findings suggest that~, rather than the concentration of a particular electron acceptor may be the main factor determining the level of cytochrome synthesis. It therefore seems unlikely that oxygen is directly involved at the molecular level as a regulator of cytochrome synthesis and that the low dissolved oxygen values at which maximal cytochrome levels have been frequently recorded are at the most an indicator of some other factor (possibly Eh} to which the cell is responding. The greatly increased values of Qo2 at steady states where the dissolved oxygen was controlled at high values (235 µMand 400 µM} are independent of any of the other parameters :measured. Cytochrome concentra tions do not rise, the value of cytochrome A showing a slight fall at 400 µM, nor is there any increase in the cell mass produced. In addition, the value of QG does not increase, suggesting that substrates ad.di tional to glucose are being oxidised. These could co.me from the yeast extract and peptone which are constituents of the medium. Certainly it seems that much of the oxygen being taken up is not being utilised for oxidative phosphory- lation. The values ofµ( )/µ( (Table 3.2, column for these ea1 c exp. l 5) high oxygen steady states reveal a vast discrepancy between the growth rate and that expected if all of the oxygen taken up was being applied to oxidative phosphorylation and growth with the maximum efficiency. Some of the excess oxygen uptake may be due to fixation by an oxidase system; Babij, Moss and Ralph (1968) have shown increasing unsaturation of fats in C. utilis with increasing values of dissolved oxygen. It is therefore possible that the lipids and perhaps many other components of the cells are at a much higher redox potential than at lower oxygen levels. Gundersen (1966) has reported a doubling of oxygen uptake by Nitrocystis oceanus grown at ~n oxygen tension of 684 mm Hg compared with cultures grown under air. Increased oxygen consumption with increasing oxygen tension has been observed in several chemoautotrophic bacteria grown on solid mineral salts medium (Gundersen, Carlucci and Bors tr om, 1966 l . Several workers have offered explanations of the mechanism and function of this phenomenon. Lenhoff, Nicholas and Kaplan (1956) and Rosenberger and Kogut (1958) have suggested the operation, under excess oxygen conditions of terminal oxidation pathways alternative to the cytochrome chain in Pseudomonas. There have been several suggestions that in Azotobacter, high oxygen uptakqunder excess oxygen conditions functions as a protective mechanism in order to maintain a low Eii_ in the vicinity of the nitrogen fixing enzymes (Philips and Johnson, 1961; Parker and Scutt ,1960; Dilworth and Parker, 1961; Kmel, et al, , 1965}, 86. A similar mechanism may well operate in yeast with the object of protecting the cells against an excessive internal Eri_ value. That a high_ internal %_ is undesirable has been illustrated by Chance, Jamieson and Coles (i965) who found that under hyperbaric o:xygen conditions (ll atmospheres of o:xygen) reduction of pyridine nucleotides was completely inhibited in suspensions of baker 1 s yeast. Thus , in an extreme o:xygen environment, normal energy metabolism, and presumably the operation of an o:xygen wasting protective mechanism would be impossible. However at o:xygen levels which are high but sub-toxic, as in the 235 µM and 400 µM steady states described here, such an o:xygen wasting mechanism may operate with the function of maintaining a sufficiently low intracellular %_ to permit metabolism to proceed. Exposure to high levels of o:xygen is toxic to a wide range of living forms. It is not surprising that microorganisms, in which the ability to adapt to differing environmental conditions is . posc..e~a most highly developed, pae111 a system for the removal of excess o:xygen from their environment when it approaches toxic levels. The operation of this protective system is manifested by an increased rate of OJcy'gen uptake. 4.2 BEHAVIOUR OF C. UTILIS DURING ADAPTATION TO A LOW OXYGEN ENVIRONMENT The step change from high to low o:xygen in the transient experiments described in section 3.3 is in effect a sudden limitation of o:xygen supply to a culture growing in steady state conditions of o:xygen abundance. In the two transient experiments, the subsequent changes which the cultures undergo in adapting to this imposed limitation show general similarities. However there are significant differences and these differences reflect the different degree of oxygen limitation which each step change imposed. In the first transient experiment (section 3.3.1) the change in dissolved o:x;vgen from 235 µM to a controlled value of 1.2 µM did not impose as severe a limitation on oxygen availability to the cell as did the step change in the second transient experiment (section 3.3.2) from a dissolved oxygen value of 235 µM to a low oxygen rolution rate. This is clear from a consideration of the method of control of dissolved o:x;vgen which allows the oxygen solution rate to be varied as widely as necessary, depending on the demand by the culture for o:xy gen • The major difference between the two transient states is the time which elapses a~er the step change before the appearance of fermentative metabolism. In both cases the onset of fermentation is seen when Qo2 has fallen to a very low value of approxilllate]y l m mole/g. /hr. The absence of fermentation other than when Q02 is very low is consistent with the findings from the steady state cultures (section 3.l}, In the second transient state, Q02 had fallen to a very low value within a few hours of the imposition of the step change, and fermentative energy metabolism, with its associated reduction in cell yield and increased Qc}, had appeared. From this point the rise in the rates of production of C02 and ethanol were notably slow, a period of 80 hours being required 88. before a steady state was reached. This suggests that the rates of synthesis of pyruvate decarbozylase and/or alcohol dehydrogenase were limited. A limiting factor in the synthesis of these enzymes may have been the rate of supply of carbon, or some essential co-factor, which is limited by the dilution rate. In the first transient state, however, 30 hours elapsed a~er the step change before Qo2 had reached a level of l m mole/g./hr. Nevertheless from this point the gradual rise in the rate of fermentative metabolism, together with its associated effects on cell yield and Qa, followed the course seen in the second transient state, although some fluctuations occurred and a longer period of time elapsed before a steady state was reached. The factors which are of particular interest are those governing the time course of Qo2 in the first transient state, particularly over the first 30 hours. The initial fall in Q02 was to a relatively high value of approximately 10 m moles/g4ir and only after 30 hr did it fall to the value expected at a controlled dissolved OJcy"gen value of 1.2 µM. During the initial steady state and over the period for which Qo2 remained at about 10 m moles/g./hr, the value of QG was constant. As has been discussed above, the high Qo2 at the steady state at 235 µM does not represent an increased turnover of glucose but is probably partly explained by utilisation of other orldisable substrates in the medium. Following the step change there seems to have been a temporary continuance of this situation at a decreased but still relatively high Q02 value, the maintenance of which was possible by virtue of the :method of control of dissolved o:x;ygen. Although this situation was not the final steady state, it did persist for 20 hours. This period of time mey have been required for the gradual disappearance of the factor which influenced the development of the high Q02 in the initial steady state, If this factor is the intra cellular redox potential, it might, however, have been expected to fall more rapidly. There may have been high levels of some intermediate or intermediates of metabolism during the initial steady state and these levels mey have persisted after the step change, the Qo2 falling only upon their depletion. Whatever the causative factor, Q02 at 30 hours had fallen to a low value and fermentation had appeared. Before this fall however Q02 underwent a rise to 15 m moles/g. /hr and this rise was associated with, and probably caused by, a rise in the cytochrome level to very high values. An important feature of the chronology of these events is that the highest cytochrome value was recorded at 29 hr, at which point fermentative metabol ism was already quite evident and dry weight had fallen quite substantially. Clearly this fleeting high cytochrome level was not related to the general pattern of change of the metabolism of the culture. It seems that the factor which was enabling Q02 to be maintained at 10 m moles/g. /hr was also a controller of cytochrome synthesis. If the fall in Qo2 to a low value indicates depletion of a metabolite, then this metabolite may be a repressor of cytochrome synthesis. If redox potential is to be implicated as a possible factor here, then the cell must be responding to its intracellular 90, ~ value since dissolved oxygen was constant from the step change. From the above considerations, then, it appears that the factor which was enabling maintenance of Q02 at 10 m moles/g./hr had disappeared at about 12 hours. The sharp fall in cytochrome concentration following the rapid rise to a maximum value has been shown (section 3.3.2} to be due at least in part to active breakdown of cytochrome by the cells, During this period, in which Q02 is low and fermentation is increasing, the breakdown of cytochrome probably indicates a reutilisation of its molecular consti tuants for synthesis of other cell components. A silllilar phenomenon of active cytochrome break.down has been observed when a chemostat culture of C. ·utilis is treated with chloramphenicol (?.P,Grey, personal co:mmunicationI. The rate of acid production, which had previously fallen, shows a rise over the period during which cytochrome concentration falls from its maximum value. This may be due to excretion of organic acids as products of cytochrome breakdown. The vast changes occurring in cytochrome levels at a constant value of dissolved oxygen during this transient period reinforce the earlier suggestion that, at least at low concentrations, oxygen does not seem to act at the molecular level as a regulator of cytochrome synthesis. Thus the notion of oJcy"gen repression of cytochrome synthesis would appear to be inaccurate. It is notable that in the first transient experiment, the new steady state, apart from the cytochrome concentration values did not correspond with the steady state which would be expected at a controlled dissolved o:xygen value of l.2 µM, on the basis of the steady state experiments dis cussed earlier. The level of fermentati ve activity was much higher in the final steady state of the first transient study than in the steady state at a dissolved oxygen value of l,5 µM (Table 3.1). This difference m.ey mean that not only the environmental conditions, but also the history of the culture in adapting to those conditions determines the physiological state of the cell. Alternatively, it rmy be that true steady state conditions had not in fact been reached at 200 hr. Certainly the final few values obtained for some of the para.meters, for example dry weight and QE, indicate that they m.ey still be changing at a very slow rate. Had measurements continued over a very much longer period, these para.meters may have changed further to reach a steady state which corresponded more closely with that expected. Clearly a complete explanation of the phenomena observed during adaptation to a low oxygen environment requires knowledge of the changes which are occurring in concentrations of metabolic intermediates and other important compounds such as pyridine nucleotides and adenosine phosphates. The study of these transient states has shown that when a steady state· culture is subjected to a sudden change in one of its environmental parameters, a smooth adaptation to a new steady state does not necessarily follow, especially when the step change is large and involves an important substrate of the organism. The technique of continuous culture has enabled 9._2. precise description of the physiological state of an organism in a particular defined environment. The extension of this technique to the study of the behaviour of an organism during transition f'rom one defined environment to another offers further information to lend support to general findings from steady state cultures, but also displeys as a function of time the operation of metabolic regulatory systems. 4. 3 DAMAGE '1'0 C • UTILIS BY OZONE Treatment with ozone in small concentrations was seen to lead to complete loss of cytochromes in c. utilis. This effect was related to a decrease in percentage viability, but a much higher degree of resistance to ozone was seen in growing cultures than in non-growing cell suspensions. This was seen in comparing a complete loss of viability in one hour in a standing culture (Table 3.11) with a maintenance of 75% viable cells in a chemostat culture where the mean time of exposure to ozone is J.O hours (Table 3.13, State 7). Although the volume of the chemostat culture was twice that of the resting suspension, the higher flow rate of ozone into the chemostat culture together with the much more efficient mixing in the continuous culture vessel and the J.O times increase in exposure time indicate that actively growing cells are able to tolerate much more severe ozone treatment than non-growing cells. Haines (1936} observed the same phenomenon with. Bacillus coll grown in Nelson's medium. He found that when an ozone atmosphere was admitted simultaneously with inoculation, growth 23. was retarded by 4 p.p.m. of ozone and completely inhibited by 10 p.p.m. However when growth was established a concentration of ozone greater than 200 p.p.m. was required to arrest it. These concentrations of ozone are much lower than those required to affect C. utilis in the experiments described here. Also the difference between the concentrations of ozone which affect microorganisms in liquid culture and concentrations f'ound in polhted air is very large indeed. However ozone is highly reactive and decomposes readily in the presence of moisture (Alder and Hill, 1950). When consideration is taken of the wlUIIe and chemical complexity of a liquid microbial culture, it becomes apparent that when ozone is admitted in concentrations comparable with air pollution levels, all of it is likely to react with components of the culture medium so that none reaches sites in the cell; The absence of ozone in the eff'lu.ent gas from the chemostat culture indicates that even at comparatively high concentratio~, all of the ozone admitted is destroyed, either by chemical combination or decomposition, In the chemostat culture, the progressive decrease in percentage viability which was seen with increasing levels of ozone dosage was not accompanied by a decrease in Q02 • The value of Q02 rose in fact, and reached very high levels during the decline of the culture in state 9 (Table 3,13). There was however a decrease in QC02 with -viability, suggesting that much of the o:xygen being consumed was not used for reoxidation of reduced coenzymes produced by operation of the TCA cycle. Giese and Christensen (1954} passed ozone through a suspension of S. cerevisiae in phosphate buffer and measured OJcy"gen uptake in the presence of glucose or galactose. They found that ozone caused a decrease in overall o:xygen uptake but that this correlated with decreased viability. They concluded that an "all-or none" effect was operating and that while there was no uptake of o:xygen by non-viable cells, the viable cells in the ozone-treated suspension continued to respire at an unchanged rate. However in the more rigorous conditions of the chemostat culture described here,steady states have been maintained (Table3,l3 states 7, 8} which show that o:xygen uptake relative to dry weight does increase with increasing ozone dosage without a significant change in viability. This steady state rise in Qo2 could be a similar phenomenon to that seen in steady state cultures at high values of dissolved OJ!ygen. Although the aeration conditions of the culture make it unlikely that the value of dissolved o:xygen was very high, the presence o:t;ozone may well have increased the redox potential of the medium in the culture -vessel to a level greater than that which might be expected at this o:xygen solution rate. As considered earlier in relation to the high o:xygen steady states, an increased Q02 under such conditions probably indicates the operation of an o:xygen-wasting protective mechanism. Ozone has been shown to stimulate respiration rate in photosynthetic microorganisms and plant tissues. De Koning and Jegier (1968a) passed 130 ml/min of air containing l p.p,m. of ozone through a 5 ml suspension of the alga Euglena gryeilis and found a decrease in photosynthesis and an 2.5. increase in respiration, Similar findings have been reported f'or pinto bean leaves and citrus fruit (Todd 1956, 19581, leaves of' the tobacco plant (Macdowall, 1965 l and the small aquatic pant LeIIltla minor (Erickson and Wedding, 1965 l. Inhibition of respiration was however demonstrated by Freebairn (19571 when suspensions of mitochondria from various sources were exposed to ozone, During the decline of the chemostat culture (Table 3.13, state 91, the very high Q~ values recorded were probably related, to oxidation of the material of non-viable cells, which made up practically all of the culture, However there was an associated increase in QG, which was no doubt a result of the appearance of' significant levels of glucose in the culture vessel. 'lhe Qo2 of the viable cells had therefore probably increased to some degi:-ee, but the magnitude of this is impossible to determine. Evidence from electron microscopy shows that the internal structure of the cells is disrupted under severe ozone treatment, Giese and Christensen (1954} studied the effect of ozone on cells of ParaJD.ecium stained with neutral red. 'Ihey found that the stain was not decolorised by ozone until advanced cell daJD.age had occurred, indicating that the initial effect of ozone is at the surface only. Their suggestion that the gi:-e at reactivity of' ozone results in oxidation of materials at the cell surface before ozone enters the cell, seems reasonable. It is likely that in the experiments described here there is little penetration of ozone into the yeast cells 9_6. before the outer layers of the cell, including the cell membrane, have been irreversibly destroyed by ozone. As discussed above, the observed stimulation of Q02 ma:y be a reaction by the cell to an increased redox potential rather than to the presence of molecular ozone inside the cell. The lack of evidence for 1ll1ltagenic action by ozone is explained by similar reasoning. If ozone at low concentration reacts almost entirely at or near the surface of the cell, the chance that molecular ozone would come in contact with the DNA of the cell are remote. Only at concentrations which cause gr-ass disruption of the cell structure would ozone reach the cell's genetic material, but in these conditions the cell would have lost its viability. In short, the very strong oxidising properties of ozone do not permit its selective action as a mutagen. However there is evidence for action of ozone in causing chromoscme aberrations in plant cells. Fetner (1958) treated dividing root meristem cells with ozone for 60 min and found 42% of the subsequent anaphases to be abnormal. 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Pirt, Eds., Cambridge University Press, p. 161.. WIMPENNY, J, W, T. , R.ANLETT, M, R. and GRAY, C, T, (1963}, Biochim. biophys, --Acta, 73, 170. WISEMAN, G,M,, VIOLAGO, F,C,, ROBERTS, E, and PENN, I. (1966), Can, J, Mi crob:tol, , g_, 521, YCAS, M. ana. DRABKIN, D.L. (1957}, J, biol. Chem, 224, 921, ZoBELL, C,E, ana. RITTLE, L,L. (1967}, Can,-,J, Microbiol., 13, l3ll, ... (~l APPENDIX· I EQUATIONS USED IN CALCULATION OF "Q" VALUES l. SYMBOLS f = flow rate of medium into culture vessel (ml/hr). V = volume of culture vessel (1.}. D = dilution. . rate = f/V (hr-1 }. X = dry weight of culture (g,/1,). = flow rate of 2N NaOH into the culture vessel (ml/hr), f alk. = concentration of glucose in the medium reservoir (% w/v 1. s = concentration of glucose in the culture supernatant(% w/v). E = concentration of ethanol in the culture supernatant (% v/v}. = flow rate of O)o/gen into the culture (ml/min). = flow rate of air into the culture (ml/min). = flow rate of nitrogen into the culture (ml/min). = concentration of o~gen in effluent gas (% v/v), = concentration of carbon dioxide in effluent gas (% v/vl. = concentration of nitrogen in effluent gas (% v /v l , 2. .Qa Rate of glucose = Rate of ad.di tion Rate of loss utilisation in medium by washout. Cnl = ""' g/hr 100 100 (f x ~l - S(f + fal.k,I 55,5 m moles/g. dry wt/hr. 100 x x x V 3. ~ A concentration of 0.20% (v/vl of ethanol in the medium reservoir is assumed. Specific gravity of ethanol= 0,79, Rate of production = Rate of loss f'rom Rate of input in of ethanol culture by washout medium 0,79E (f + f 1k 1 ______0,2 X 0,79 (f + f a_lk_. ) /hr = a • 100 100 g. 0. 79 ( f + f 1k l (E - 0. 2} = a • g,/hr 100 . 0,79 (f + falk.,) (E - 0.2} 21,74 . . ~ = m moles/ g, /hr • 100 x x x V Rate of o~gen = Nett flow of Nett flow of oxygen utilisation oxygen into from culture. culture O, 791 f . + fN l = f 02 + o. 209 f . P 02 C a.J.r 2 air ml/min 100 - (P02 + Pco2} = 60 x Rate of OJcy'gen utilisation m moles/g./hr. 22.4 X X XV (xii} 5. %02 Rate of co (0.791 f. + fN 2 air 2 l Pco2 = ml/min. production 100 - (Po2 + Pco2l 60 x Rate of C02 production Qco2 = m moles/ g. /hr. 22.4 xxxV 6. Acid production Rate of acid = ml N NaOH/ g. /hr, production xxV (xiii} APPENDIX II SAMPLE CALCULATION OF PREDICTED CYTOCHROME A AND DRY WEIGHT VALUES IN TABLE 3.6. From Table 3,6, the dry weight value at l.75 hr is 6.61 g./1. The contribution to this value from cells present at time zero is given by -Dt = .x e ~ -0 where x = 6 .61 g. /1., -0 -l D = O.l hr , and t = 1.75 hr 6.6l X ,;(0.l X l.75} .. ~ = = 6,34 g. /1. The fraction of the original cells present at l.75 hr is therefore 6.34 b.bl or 0.96. Thus, at l.75 hr the culture is composed of 96% original cells with a cytochrome A value of 91.5 and 4% new cells with a cytochrome A value of O. The predicted c-y!Dchrome A value of the culture at 1.75 hr is therefore 91.5 x 0,96 or §.L.2.. Cx:tyl PUBLICATION SUBMITTED IN SUPPORT OF THESIS "The Response by Microorganisms to Steady State Growth in Controlled Concentrations of 0Jcy"gen and Glucose. I. Candida utilis 11 • F.J. MOSS, PAMELA A.D. RICKARD, G.A. BEECH and F.E. BUSH. Biotechnology and Bioengineering, ll (1969) in press.