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,<CJI'ILIS BY OZONE 92 5.