Bioprospecting Surfactants Produced by Pseudomonas spp. Isolated from Soil for Potential Application in Biotechnology A thesis submitted for the degree of Doctor of Philosophy (PhD) by Kamaluddeen Kabir BSc, MSc. School of Science, Engineering and Technology, Abertay University. April, 2017 Preliminary pages K. Kabir Page ii Preliminary pages Declarations I, Kamaluddeen Kabir, hereby certify that this thesis submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy (PhD), Abertay University, is wholly my own work unless otherwise referenced or acknowledged. This work has not been submitted for any other qualification at any other academic institution. 02/08/17 …………………………………………………. ………………………………………………….. Candidate’s Signature Date K. Kabir Page iii Preliminary pages Acknowledgements I would like to express my sincere gratitude to my supervisor, Dr Andrew Spiers. He has continuously supported me during my PhD programme at Abertay University, and his patience, motivation and advice on ideas have been a source of inspiration to me. This thesis would not have been accomplished without his intellectual input. His supervision has guided me throughout my research and writing of this thesis, and I cannot imagine having a better supervisor or mentor. My immense gratitude also goes to my second supervisor, Dr Yusuf Deeni, for his intuitive comments and support. His questions and input truly helped me to widen my research and look at it from different angles. Even with his tight schedule, he took the time to go through my entire thesis and for this I have nothing to say but, ‘JazakAllah khair’. My sincere gratitude also goes to Zuzana Spiers for her help during the Sample collection and for making my stay in Dundee a memorable one. I also hereby extend my gratitude to the Head of Graduate School, Dr Nia White and her assistance Miss Wendy Robb for their support and guidance. I remain indebted to my parents Alhaji Kabir and Hajiya Halima for their extensive role in my upbringing and education. Their continuous support in any times of difficulty cannot be under-estimated: they will forever be precious in my heart. My gratitude goes to my step mother, Hajiya Sa’adatu and to my brothers and sisters Khadija, Zaharaddeen, Nafisa, Nura, Aisha, Saratu and Pharm. Muhammad, and to Hayatu, Sadam, Binta, Farida, Jalaluddeen, Lubabatu, Tajuddeen, Ahmed and Kamaraddeen, as well as to all those I have not mentioned but who are not forgotten. To my uncles, aunts, nephews and nieces, thank you for all your prayers and concern. K. Kabir Page iv Preliminary pages To my wife Aisha and daughter Saratu, thank you for your continuous understanding and motivation. You gave me joy when I was tired and kept me on track - thank you. And to my in-laws Muhammad Jamil Yusuf and family. A final thanks also goes to Uncle Ismail and family, thank you for your kindness, care and support in making life easier in Dundee. I would like to thank my colleagues at the Graduate School for their interesting discussions and all the fun we have had over the last few years. In particular, I would like to acknowledge Ibrahim Kankia and Raji. A big thanks is owed to my friends here in the United Kingdom including Solomon Anjide, Musa Ladan, Aliyu and Armaya’u. My conscience would not rest if I did not acknowledge my friends back home, Abdullateef, Nura, Umar Zango, Auwal Dansharif, Abubakar, Jamil Bashir, Baba Bala, Aminu Dankanjiba, Ibrahim Barguy, and others too many to name for their spiritual and verbal support throughout my stay at Abertay. A special thanks goes to Prof. Mu’utasim Ibrahim and family and to Engr. Muttaqha Rabe Darma for their part in supporting me in attaining this level of my career; Allah will surely reward you. I also hereby extend my gratitude to Malik Anas, Dr Dikko Suleiman and Family, Alhaji Galadima and Hajiya Jummai of PTDF and Muhammad Ahmad and Hayatu Ruma of Umaru Musa Yar’adua University. Last but not least, I would like to acknowledge my sponsors Umaru Musa Yar’adua University Katsina (UMYU) and the Tertiary Education Trust Fund (TETFUND) for sponsoring my research at Abertay University, Dundee, United Kingdom. My thanks also to the other societies that sponsored some of my conference attendance. K. Kabir Page v Preliminary pages Dedication This thesis is dedicated to my beloved parents, Alhaji Kabir Mohammed and Hajiya Halima Ali Yar’adua, for all their help, caring and encouragement, and for raising me and giving me hope in life. May Allah continue to guide and protect them in all their doings. And to my wife Aisha and daughter Saratu Kamaluddeen, I dedicate this to you both for being my source of happiness. K. Kabir Page vi Preliminary pages Abstract Bacteria produce a range of surface-active compounds called biosurfactants that reduce the surface tension of liquid and exhibit different oil-water behaviours. These are used in various biotechnological applications including agriculture, cosmetics, medical and food. A recent study has predicted a limit to bacterial surface tension-reducing ability. If this limit exists, it has strong negative consequences in surveys for more active compounds. In this work, the aim is to (i) investigate this prediction more robustly by using chemical media and (ii) study the diversity amongst the best-performing surfactants produced by Pseudomonas spp. with the intention of finding novel surfactants that could be used in different biotechnological applications. A total of 251 Pseudomonas spp. were isolated from soil. Strains were first screened for liquid surface tension-reducing ability (LSTRA) using qualitative drop-collapse assay before quantitative surface tension measurement. Of the 58 LSTRA strains, only 46 significantly reduced the surface tension of sterile media. Individual Distribution Identification (IDI) analysis was used to determine the predicted limit for surfactant activity in KB* and M9Glu media, and results were found to be in agreement with earlier studies. To investigate the chemical structural diversity amongst the best performing surfactants, a collection of 25 key strains producing a limited range of very low surface tension in liquid culture media (~24 – 26 mN/m) were examined. Initial phenotypic characterisation including biochemical, metabolic profiling and 16S rDNA sequencing confirmed strains were a diverse collection of Pseudomonas spp.. A series of behaviour assays including emulsion formation, foam stabilisation and oil displacement assays to investigate behavioural diversity among surfactants expressed by the key strains were then undertaken. For the oil displacement, diesel, mineral, vegetable, and used lubricating oils were tested with the underlying aqueous layer containing 0 or 200 mM NaCl at pH 6.0 or 8.0 to reflect a range of biotechnological applications and conditions. Analysis of variance of the emulsion indices, foam stabilisation and oil displacement data showed significant difference in surfactant behaviour among the key surfactant-expressing strains (P < 0.001). Moreover, Hierarchical Cluster Analysis (HCA) was used to produce a constellation dendrogram in which isolates were grouped according to similarities in phenotype and surfactant behaviour. Critically, this resulted in more groups (≥ 5 groups) than could be explained by statistically significant differences in mean surface tensions (previously determined by ANOVA and Tukey-Kramer HSD, alpha = 0.05). These findings provide strong evidence that the key strains were expressing structurally more than one type of surfactant with differing air-water and oil-water behaviours. Similarly, in vitro surfactant characterisation within a range of pH and salt concentrations confirmed diversity among strains (P < 0.001). Investigating surfactant potential by a two-way behaviour cluster dendrogram resulted in more diversity among oil types than the conditions used. These findings indicate that bioprospecting surfactants by screening only the more active compounds is likely to reveal a range of functionalities. K. Kabir Page vii Preliminary pages Table of Contents Certification ................................................................... Error! Bookmark not defined. Declarations ................................................................................................................ iii Acknowledgements ..................................................................................................... iv Dedication ................................................................................................................... vi Abstract .......................................................................................................................vii Table of Contents ....................................................................................................... viii List of Figures ............................................................................................................. xiii List of tables ................................................................................................................ xv Definitions ................................................................................................................... xv Chapter 1 Introduction ................................................................................................ 1 Preface ......................................................................................................................... 1 Chapter 1 Introduction ................................................................................................