Lipid Humification by Soil Clays by ADRIAN RICHARD ADAMS Dissertation presented for the degree of DOCTOR OF PHILOSOPHY in the Faculty of AgriSciences at Stellenbosch University Supervisor: Dr C.E. Clarke Co-supervisor: Dr A.G. Hardie April 2019 The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. Stellenbosch University https://scholar.sun.ac.za Declaration By submitting this dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: April 2019 Copyright © 2019 Stellenbosch University. ALL RIGHTS RESERVED. Stellenbosch University https://scholar.sun.ac.za “You are young yet, my friend,” replied my host, “but the time will arrive when you will learn to judge for yourself of what is going on in the world, without trusting to the gossip of others.” “Believe nothing you hear, and only one half that you see.” —Edgar Allan Poe “The system of Doctor Tarr and Professor Fether” (Graham's Magazine, vol. XXVIII, no. 5, November 1845) Stellenbosch University https://scholar.sun.ac.za Abstract Lipid Humification by Soil Clays A.R. Adams Department of Soil Science Stellenbosch University Private Bag X1, Matieland 7602, South Africa Dissertation: Ph.D. (Soil Science) April 2019 We studied three aspects of the natural polymerisation (humification) of lipids by soil clays—namely, the products formed, reaction mechanism and kinet- ics—at environmental temperatures (c. 20–50±C). Various clays were reacted with oleic acid (our chosen model lipid). The Mn-oxide birnessite was the most reactive toward oleic acid, polymerising it into quasi-solid polyesters. A probing of the birnessite-oleic acid reaction mechanism revealed that the formation of a surface exchange complex between oleic acid carboxyl groups and birnessite surface sites ( Mn3+/ Mn4+) is a crucial first step of the reac- ´ ´ tion. Subsequent chelation and one-electron reduction of Mn3+ to Mn2+ forms radical oleic acid species which couple and thereby polymerise. Kinetic studies revealed that the birnessite-oleic acid reaction was near- linearly dependent on birnessite mass-loading (rate order 0.75) but virtually » independent of birnessite surface pH (rate order 0.2). A determined activa- » tion energy (E ) for the reaction of 12.8 4.2 kJ mol 1 revealed that it is ener- a § ¡ getically more spontaneous than the usual autoxidation pathways. These findings broaden our understanding of the role soil clays play in lipid humification in soils. Keywords: humification, lipids, soil minerals, polyesters, complexation, RGB colour analysis — iii — Stellenbosch University https://scholar.sun.ac.za Uittreksel Lipied Humifikasie deur Grond Kleie “Lipid Humification by Soil Clays” A.R. Adams Departement Grondkunde Stellenbosch Universiteit Privaatsak X1, Matieland 7602, Suid-Afrika Proefskrif: Ph.D. (Grondkunde) April 2019 Ons het drie aspekte van die natuurlike polimerisasie (humifikasie) van lipiede deur grond kleie bestudeer—naamlik, die produkte gevorm, reaksie meganisme en kinetika—teen omgewings-temperature (ong. 20–50±C). Verskeie kleie is met oleïen suur (ons gekose model lipied) gereageer. Die Mn-oksied birnessiet was die mees reaktief teenoor oleïen suur, deur dit te polimeriseer na kwasi- soliede poliësters. ‘n Ondersoek na die birnessiet-oleïen suur reaksie meganisme toon dat die formasie van ‘n oppervlaks-uitruilingskompleks tussen oleïen suur karboksiel groepe en birnessiet oppervlaks-liggings ( Mn3+/ Mn4+) ‘n noodsaaklike ´ ´ eerste stap van die reaksie is. Gevolglike chelasie en een-elektron reduksie van Mn3+ na Mn2+ form radikaal oleïen suur spesies wat koppel en daardeur poli- meriseer. Kinetika studies toon dat die birnessiet-oleïen suur reaksie naby-liniêr af- hanklik is op birnessiet massa-lading (tempo orde 0.75) maar so te sê onaf- » hanklik is van birnessiet oppervlaks-pH (tempo orde 0.2). ‘n Bepaalde akti- » verings-energie (E ) vir dié reaksie van 12.8 4.2 kJ mol 1 toon dat dit energiek a § ¡ meer spontaan is as die gewone outoksidasie roetes. Hierdie bevindings verbreed ons begrip van die rol wat grond kleie in lipied humifikasie in gronde speel. Sleutelwoorde: humifikasie, lipiede, grond-minerale, poliësters, kompleksering, RGB kleur analiese — iv — Stellenbosch University https://scholar.sun.ac.za Acknowledgements I would like to thank my supervisors Doctor Cathy Clarke and Doctor Ailsa Hardie for their support, encouragement, persistence and patience throughout this study. Tackling a new scientific endeavour is never easy and knowing that I was not walking this path alone helped immensely. Their contributions are immeasurable. A debt of gratitude is also due to the Department of Soil Science, my home from home for over six years—to all my colleagues, staff members and friends during this time—it was a truly engaging, enlightening and fun period. I am eternally grateful for the amazing opportunity to teach and mentor undergrad- uate soil science students, especially during their rock and mineral identifica- tion practicals. Words could never fully express how much of a rewarding experience that was for me, and how much I grew personally from it. Similar holds true for our annual faculty open days, the opportunity to engage with the public and “sell” our “product” was a lot of fun. On the university campus, I would like to thank various departments, indi- viduals and entities for all their contributions to this study, be it analytical services, consumables or just general great advice and friendship. These include the Departments of Chemistry (Dr Paul Verhoeven, Prof. Len Barbour, Mr Wesley Feldmann, Dr Leigh Loots and Mrs Peta Steyn), Process Engineering (Mrs Hanlie Botha), Plant Pathology (Mrs Anria Pretorius and Mrs Tammy Jensen), Physics (Prof. Paul Papka), Botany and Zoology (Dr Aleysia Kleinert and Prof. Alex Valentine), Earth Sciences (Prof. Alakendra Roychoudhury) as well as the Central Analytic Facility (CAF; Mr Lucky Mokwena, Dr Marietjie Stander, Mr Malcolm Taylor, Dr Jaco Brand, Mrs Riana Rossouw, Mrs Charney Anderson Small and Mr Herschel Achilles). I would also like to thank the uni- versity for financial support. Outside the university I wish to thank the Inkaba yeAfrica programme and National Research Foundation (NRF) for generous financial support and scholarships. Further thanks go to Dr Remy Butcher and Mr Zakhelumuzi Khumalo for XRD analyses at the particle accelerator wing of iThemba Labs near Cape Town. Last, but certainly not least, I would like to thank my loving and tirelessly dedicated parents and Creator, for the years and years of endless support and love—for always standing by me no matter what it is I embark on—I truly could never ever have asked for more. — v — Stellenbosch University https://scholar.sun.ac.za Contents Acknowledgements v 1 Introduction 1 1.1 Rationale behind this study . 2 1.2 Aims and objectives . 2 1.3 Format of this dissertation . 3 2 Literature Review 5 2.1 Global carbon pools and fluxes . 5 2.2 Humic substances . 9 2.3 Humification . 10 2.4 Organic matter recalcitrance . 15 2.5 Lipid chemistry . 17 2.6 The properties and chemistry of soil clays . 37 2.7 Summary . 44 3 Lipid-clay interactions: screening mineral reactivity and products formed 47 3.1 Introduction . 47 3.2 Materials and methods . 49 3.3 Results and discussion . 53 3.4 Summary and conclusions . 78 4 Lipid-clay interactions: reaction mechanisms 81 4.1 Introduction . 82 4.2 Materials and methods . 83 4.3 Results and discussion . 86 4.4 Reaction mechanism . 99 4.5 Summary and conclusions . 109 5 Kinetics of the lipid-clay reaction 111 5.1 Introduction . 111 5.2 Materials and methods . 114 5.3 Results and discussion . 117 5.4 Summary and conclusions . 130 — vi — Stellenbosch University https://scholar.sun.ac.za 6 Environmental significance and implications 131 6.1 Introduction . 131 6.2 Interactions with natural reactions and processes . 132 6.3 Environmental remediation applications . 134 7 General conclusions and future work 137 7.1 General conclusions . 137 7.2 Future work . 140 References 143 Appendices A Background discussion on kinetics—rate expressions, rate constants, reaction orders and activation energy . A1 B Identifying XRD diffractograms for the clays used in this study . B1 C ATR-FTIR spectra for the clays used in this study . C1 D Calibration of the Mn3+-pyrophosphate extraction and UV-VIS quantification method . D1 E Autoxidation of oleic acid, linoleic acid and natural oils . E1 — vii — Stellenbosch University https://scholar.sun.ac.za List of Figures 1.1 Increasing data detail and downward data enrichment . 3 1 2.1 Global carbon pools (Pg C) and fluxes (Pg C a¡ ) . 6 2.2 Visual illustration of humification (polyphenols as example) . 11 2.3 The three major humification pathways found in soils . 13 2.4 Three of the most important isomers of linoleic acid . 24 2.5 Radical scavengers and pigments commonly found in olive oil . 25 2.6 The acid and alkaline hydrolysis of the triglyceride triolein . 26 2.7 The transesterification/methylation reaction to FAMEs . 27 2.8 The lipid peroxidation reaction mechanism . 33 2.9 Secondary oxidation products of lipid peroxidation . 34 2.10 Peroxidative polymerisation of FFAs and triglycerides . 35 2.11 Non-peroxidative polymer and ring products . 35 3.1 The chemical structure of the oleic acid molecule . 48 3.2 Visual changes to oleic acid-clay mixtures over 6 months . 54 3.3 Colour changes of the birnessite pyrophosphate extract . 59 3.4 Colour changes of the EDTA extracts of Fe-clays .