Enhanced Bioprocessing of Viable Nematodes John Matthew Young A thesis submitted for the degree of Doctor of Philosophy to the University of London The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Torrington Place London WCIE 7JE September 1998 ProQuest Number: U641996 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U641996 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Production of nematode-based pesticides involves the recovery of a viable nematode life stage known as an infective juvenile (IJ) from fermentation broth. The objective of this project was to identify large-scale methods for separating viable Us from waste components of liquid nematode cultures. These investigations were conducted using cultures of three nematode species; Steinernema feltiae, Heterorhabditis megidis and Phasmarhabditis hermaphrodita. Culture composition was determined so that the separation problem could be defined. Waste components to be separated from Us included non-U life stages (Jls, J2s, J4s, adults), dead nematodes, nematode debris, spent media and the nematodes’ associated bacteria. To identify potential methods of separation, physical properties (solids content, U ‘shear’ stability, viscosity, component size, component density) of cultures were measured. Measurements indicated that a low ‘shear’ recovery process is required and that solid waste can be separated using differences in component size, density and settling rate as a basis. Predictions of U settling velocity, using property measurements, suggested that culture liquid should be separated by centrifugation as opposed to gravity settling. Separation characteristics were determined and verified using small-scale tests. Culture liquid could be separated by centrifugation but not by dead-end filtration. Solid waste was separated by flotation, settling, and sieving. Large-scale gravity settling, centrifugal settling and centrifugal flotation operations were evaluated using P. hermaphrodita. Centrifugal flotation was the most efficient operation. When culture solids were passed through a bowl centrifuge, after increasing continuous phase density using sucrose solution, solid waste was separated by flotation. There was no measurable loss of product. Separation efficiency was greater than that of comparable recovery procedures described in the literature. An integrated recovery process design is suggested, and involves centrifugal classification followed by centrifugal flotation. Density-adjusting medium used for flotation is recovered by filtration and evaporation and is then recycled. Acknowledgements I am indebted to the following people, from the Advanced Centre for Biochemical Engineering at University College London, for their valuable help. My academic supervisor, Peter Dunnill, for his continual support and enthusiastic supervision throughout the project. Clive Osborne and Stuart Pope for assistance with large-scale separation experiments at the Advanced Centre. To permit nematode cultivation, they also commissioned an airlift reactor that kept contaminants out during every fermentation. Technicians in the Engineering Workshop serviced and prepared numerous items of experimental equipment. They provided a rapid service and workmanship was of a high standard. Mike Hoare and Andrew Ison gave useful feedback on the work that I presented. I also thank Parvis Shamlou for helpful discussions about characterisation of shear stability. I thank all individuals from Microbio Ltd who provided assistance. Jeremy Pearce provided me with valuable supervision at all stages of the project. During the initial stages John Godliman and Mark Shepherd taught me about the practicalities of nematode production at the manufacturing facility in Littlehampton. They also provided much valuable advice and useful discussions about nematode processing. Susan Robinson from Procter and Gamble, and Bob Davies from Foster Wheeler gave up much of their spare time to read a draft copy of the thesis and they provided comments that were particularly helpful. I also thank Lynn Peacock from Zeneca for proof reading the final version of the thesis. The Biotechnology and Biological Sciences Research Council (BBSRC) sponsored this research. The Council’s support and the collaboration of Microbio Ltd. with The Advanced Centre is gratefully acknowledged. Ill The true end of knowledge is the discovery of all operations and possibilities of operations from immortality, if it were possible, to the meanest mechanical practice. Francis Bacon 1561-1626: Valerius Terminus IV Brief Contents ABSTRACT...................................................................................................... ii 1. INTRODUCTION 1 2. MATERIALS AND METHODS 32 3. PURPOSE OF THE RECOVERY PROCEDURE..................................... 52 4. CULTURE PROPERTIES AND IDENTIFICATION OF POTENTIAL RECOVERY METHODS 58 5. DETERMINATION AND VERIFICATION OF SEPARATION CHARACTERISTICS 82 6. EVALUATION OF LARGE-SCALE RECOVERY OPERATIONS AND PROCESS INTEGRATION 110 7. CONCLUSIONS ................................................................................................ 125 APPENDICES...................................................................................................128 NOMENCLATURE......................................................................................... 162 GLOSSARY.......................................................................................................164 REFERENCES..................................................................................................167 Contents Abstract ................................................................................................................... ii Acknowledgements.................................................................................................. iii Brief Contents........................................................................................................ v Contents................................................................................................................... vi List of Figures......................................................................................................... xii List of Tables.......................................................................................................... xiv 1. INTRODUCTION............................................................................................ 1 1.1 Project significance and goal ........................................................................... 1 1.2 Biology of nematodes used as pesticides.......................................................... 2 1.2.1 Definition of a nematode.................................................................... 2 1.2.2 General morphology and structure.................................................. 2 1.2.3 Taxonomy.......................................................................................... 3 1.2.4 Life cycle and reproduction.............................................................. 4 1.2.5 The infective life stage ...................................................................... 7 1.3 Review of methods for the production of nematode-based pesticides 8 1.3.1 Process overview................................................................................ 8 1.3.2 Maintenance of nematode and bacteria isolates .............................. 9 1.3.3 Nematode cultivation ......................................................................... 10 1.3.3.1 Methods of cultivation...................................................................... 10 1.3.3.2 Commercial production.................................................................... 13 1.3.4 Recovery of nematodes from liquid cultures.................................. 14 1.3.5 Nematode storage .............................................................................. 15 1.3.6 Product formulation.......................................................................... 17 1.3.7 Conclusions from review and project objective.............................. 18 1.4 Research strategy .............................................................................................. 19 1.4.1 Introduction to the design of a recovery process............................. 19 1.4.2 Defining the purpose of the recovery procedure............................. 19 VI 1.4.3 Measurement of culture properties and identification of recovery methods........................................................................................ 20 1.4.3.1 Solids content.................................................................................... 22 1.4.3.2 Infective juvenile shear stability ....................................................... 22 1.4.3.3 Viscosity.......................................................................................... 24 1.4.3.4 Particle size .....................................................................................