Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and

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1 Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

2 METABOLISM OF LACTIC ACID BACTERIA A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand. Terence David THOMAS 1968 M Alillifillill Ilifll Tllllllliiif Y

3 METABOLISM OF LACTIC ACID BACTERIA A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand. Terence David THOMAS 1968

4 N... ABSTRACT Streptococcus lactis organisms were grown in lactoselimited batch culture and the survival characteristics of washed organisms were examined at the growth temperature. Washed suspensions had high initial viabilities ()99%) which were maintained for varying periods depending on the presence of certain added materials in the buffer and the 2+ conditions of incubation. Added Mg markedly prolonged survival, while high bacterial concentrations also extended survival times, probably because Mg 2+ was excreted by the bacteria. Surviving organisms in some conditions showed prolonged division lags, especially in the absence of Mg 2+. Addition of trace amounts of EDTA decreased the death rate by removing toxic cation impurities, while the buffer salt concentration had little effect on survival within wide limits. The optimum ph value for survival was near 7.0 and survival times increased considerably at lower temperatures. Agitation and aeration tended to decrease survival and the death rate was not influenced by the phase of growth at which the organisms were harvested from a lactose-limiting medium. Addition of casamino acids increased survival times mar k e dl y ln. th e presence of Mg 2+ " arglnlne was a 1 mos t as effective as the complete mixture of amino acids while other individual amino acids tested gave only slight increases in survival times. Fermentable carbohydrates accelerated death of starved organisms irrespective of the growth phase from which they were harvested and of the limiting nutrient; by addition of Mg the accelerated death rate was reduced Glucose metabolism proceeded at a much faster rate than arginine metabolism, theoretically producing about 7.5 times as much ATP. This rapid generation of ATP may be responsible for the more rapid

5 iii. death rates with added carbohydrates. Arginine substantially reduced the lethal effect of adverse ph values and suppressed the leakage of free intracellular amino acids into the external medium. Survival studies were followed by an investigation of the changes which took place in starved or ganisms and their relation to survival. No polyglucose or poly- -hydroxybutyrat was detected and starved organisms had a negligible respiration rate. Soluble protein was released from viable organisms. into the suspending buffer and the intracellular free amino acid pool declined steadily with the components appearing in the suspending buffer; a net increase in the total amount of free amino acid indicated some protein hydrolysis. Chloramphenicol reduced the death rates in some environments, possibly by suppressing protein degradation. RNA was hydrolysed with the release of u.v.-absorbing bases and ribose from the organisms. Conditions which promoted rapid RNA breakdown also produced rapid death rates and long cell division lags in surviving organisms. There was no appreciable degradation of carbohydrate or DNA. After 28 hr. starvation in buffer containing Mg 2+, the bacterial dry wt. decreased by 26%; loss of RNA, protein and free amino acids accounted for 10.3%, 7.3% and 2.7% of the total bacterial mass loss. The products of polymer hydrolysis appeared to be released in an undegraded form into the external buffer and there was no appreciable formation of lactate, ammonia or volatile fatty acids possibly indicating the absence of any important endogenous energy sources. Protein synthesis, determined by the incorporation of valine_ 14 C into TCA-insoluble material, was barely detectable when organisms were starved in buffer containing Mg 2+. Addition of an energy source allowed limited protein synthesis while glucose produced a much highe r rate of valine_ 14 C uptake and incorporation than arginine.

6 iv. Although arginine prolonged survival this was not due to the limited protein synthesis which took place. The survival capacity of starve d organisms could be correlated with the ability to synthesize protein which in turn may be correlated with RNA stability. A new method was developed for the assay of glycolytic activity in microorganisms. Organisms were incubated with glucose-u- 14 C and samples removed at intervals. Samples were chromatographed on DBAE-cellulose paper strips in deionized water which separated the radioactive anionic products of glycolysis (lactate, acetate and formate) from the unfermented glucose. The activity of the two fractions was then determined by liquid scintillation counting. The glycolytic activity of starved organisms declined steadily and was not correlated with survival. Phospholipid was broken down on prolonged starvation and the permeability properties of the organism were gradually lost. Addition of spermine gave enhanced survival and suppressed the release of u.v.-absorbing material. Lactic dehydrogenase and DNA were released as the death rate increased in buffer containing Mg2 + and eventually, well after death, cell lysis occurred. Electron micrographs indicated that addition of amino acids maintained cell structures for a much longer period and in this system cell lysis occurred as the death rate increased. It was concluded that the death rate of starved S. lactis organisms in phosphate buffer was partly dependent on the presence of Mg2 +, which probably acted by promoting polymer stability, particularly that of RNA. In this environment, a suitable exogenous energy source further enhanced survival which may ultimately be a function of cell wall and membrane stability.

7 v. ACKNOWLEDGEMENTS The author is ind bted to the New Zealand Dairy Research Institute for providing the opportunity to undertake this investigation. Particular thanks are extended to: Professor R. D. Batt and Dr. W.A. McGillivray for their advice and encouragement throughout the course of this work; Dn R.C. Lawrence and Mr. J.G. Robertson for helpful comments on the manuscript ; Mrs. P. Lyttleton and Mr. K.I. Williamson for preparing the electron micrographs; Dr. C.R. Boswell for assisting with atomic absorption spectrometry measurements ; Mr. J.G. Dingle for assisting with amino acid analyses; Mr. R.V. Toms for the reproduction of figures and plates; Mrs. J. Crompton and Mrs. R. V. Haggett for typing the manuscript.

8 vi. PREFACE A major function of the Microbiology Department of the New Zealand Dairy Re search Institute is the maintenance of active cultures of various lactic acid bacteria for use in the cheese industry. This work is vital for the efficient functioning of one of the country' s maj or industries. The most important cheese 'starter' organisms are the lactic streptococci. This group consists of Streptococcus lactis, Streptococcus cremoris and variants. No detailed measurements have been reported on factors affecting the survival and activity of lactic streptococci, or closely related bacteria, at growth temperatures. Accordingly, it was decided to undertake such an investigation which could possibly produce results of considerable practical significance. The Introduction is d vided into two parts. Part I contains a literature review on the metabolism of lactic acid bacteria which provides a background for subsequent discussions. Part II contains a summary of the reports most relevant to studies on the survival of S. lactis. Publications to date from results presented in this thesis are entitled: 'Survival of Streptococcus lactis in starvation conditions, I. ge n. Microbiol., iq, 367, (1968), 'A new method for the assay of glycolytic activity with special reference to microorganisms', Anal. Biochem., in Press.

9 vii. CONTENTS INTRODUCTION PART I. PART II. THE METABOLISM OF LACTIC ACID BACTERIA WITH SPECIAL REFERENCE TO STREPTOCOCCUS LACTIS 1 ( i ) ( ii) ( iii) ( iv) ( v ) General characteristics Carbohydrate fermentation Metabolism of non-carbohydrate compounds Growth requirements Summary Page SURVIVAL OF VEGETATIVE MICROORGANISMS 13 ( i ) Definition of terms ( ii) Methodology ( iii) Re sults of viability measurements on microorganisms starved at the growth temperature under 'minimumstress ' conditions 17 ( a ) Effect of ionic environment 17 (b ) Effect of exogenous substrates 19 ( c ) Effect of bacterial density 2 1 ( iv) Catabolism and turnove r of cell ( v) constituents in viable, starved bacteria 23 The role of endogenous metabolism in the survival of starved bacteria AIMS OF THE PRESENT INVESTIGATION 35 METHODS 37 Microbiological procedures 37 Analytical methods 45 Glycolytic activity assay 54 Electron microscopy 62

10 viii. EXPERIMENTAL PART 1. Page SURVIVAL OF STREPTOCOCCUS LACTIS 64 Growth of the organism 64 Bacterial numbers in resuspende d systems 66 Survival in resuspende d systems 70 Effect of: EDTA 70 divalent metal ions 70 bacterial concentration 71 growth phase and media composition 72 salt concentration 72 ph value temperature 74 atmosphere and agitation 75 metabolic inhibitors 75 added carbohydrates 77 added amino acids and other growth 77 medium components Growth characteristics of survivors PART II. CHANGES IN VIABLE ORGANISMS IN STARVATION CONDITIONS ( i ) ( ii) ( iii) ( iv) Chemical studies 'Reserve' polymers 79 Oxygen uptake studies 80 Changes in bacterial protein and 80 total nitrogen Re lease of amino acids and ammonia 83 Changes in nucleic acids 87 Changes in carbohydrate 90 Changes in lipids 90 Protein synthesis in starved S. lactis 95 Metabolism of arginine and glucose 100 Changes in perme ability and ultra- 102 structure Measurement of bacterial lysis

11 ix. Page Effect of spermine General ultrastructural features of the cell Ultrastructural changes in starved cells DISCUSSION Survival of starved bacteria Survival measurement Eff ect of : Mg 2+ bacterial density added substrates temperature and ph Changes in starved organisms Polymer degradation Metabolism of arginine and glucose Changes in permeability and Conclusions ultrastructure III III REFERENCES 124