A Feasibility Study to Scale-Up The Production of Sporosarcina pasteurii, Using Industrial-Grade Reagents, For Cost-Effective In-Situ Biocementation Armstrong Ighodalo Omoregie Doctor of Philosophy (Science) September 2020 School of Chemical Engineering and Science, Faculty of Engineering, Computing and Science i A Feasibility Study to Scale-Up The Production of Sporosarcina pasteurii, Using Industrial-Grade Reagents, For Cost-Effective In-Situ Biocementation Armstrong Ighodalo Omoregie MSc. (Res.), BSc. (Biotech.) A thesis by publication submitted to the School of Chemical Engineering and Science, Faculty of Engineering, Computing and Science, Swinburne University of Technology Sarawak Campus in total fulfilment of the requirement for the degree of Doctor of Philosophy September 2020 ii ABSTRACT Biocementation is a relatively new biotechnological technology which utilizes ureolytic bacterial cells (e.g. Sporosarcina pasteurii) to stimulate insoluble biominerals (i.e. calcium carbonate) precipitation for soil improvement through ureolysis-drive microbially induced carbonate precipitation (MICP). The MICP technology serves as a suitable alternative to several chemical and mechanical methods to improve the mechanical properties of soil specifically due to its less ecological risks or environmental hazard. Soil solidification via MICP occurs when calcium carbonate is precipitated within the soil matrix, which fills the pores and cements soil particles, thereby enhancing the engineering properties such as compressive strength and permeability. MICP permits calcium carbonate precipitation to occur from dissolved calcium ions under urea hydrolysis through the catalysis of urease by ureolytic bacterial cells. The versatility of MICP technology is promising but its practicality on a commercial- scale still faces vehement economic impediments. This is because most investigations on MICP typically use expensive analytical-grade reagents (i.e. yeast extract, urea and calcium salts) which are often done under laboratory conditions to ensure quality control and reproducibility of biocementation. This has unfortunately made MICP technology susceptible to high-cost and unsuitable for field-scale implementation. The main objective of this research was to investigate a feasible economic strategy which can scale-up ureolytic bacterial cells for in-situ MICP implementation. The study from this thesis provides an essential contribution for in-situ MICP application from an economic viewpoint. To encourage future low-cost soil biocementation experiments, the use of technical-grade reagents as a replacement to costly analytical-grade reagents and a custom- built stainless steel stirred tank reactor (3 m3) to scale-up the production of ureolytic bacteria under non-sterile condition were reported. Firstly, food-grade yeast extract habitually used for bakery and cooking purposes was explored as an alternative growth substrate for cultivation of S. pasteurii cells based on biomass production, pH, urease activity and biomineralization. High maltodextrin concentration (40%) was present in the food-grade yeast extract which came with the product from the manufacturer and not deliberately added to the cultivation medium. However, the maltodextrin content did not inhibit the growth of the bacterial cells. iii Results from the effect of different media concentrations and initial pH medium showed that 15 g L−1 (w/v) and initial pH 8.5 constituted the highest biomass concentration and urease activity for the cultivation of ureolytic bacterial cells. Comparison with eight selected laboratory-grade media showed similar performance and significant reduction of bacterial cultivation cost (99.80%). Also, this thesis comprehensively studied the efficacy of using different concentrations (0.25 to 1.0 M) of technical-grade reagents when prepared in tap water and deionized water for soil biocalcification. MICP treatment of soil columns via surface percolation method under laboratory-condition was performed. The soil treatment with the inexpensive cementation reagents was compared with analytical-grade cementation reagents. The MICP aided in clogging the fine pores of sand particles after liquid cementation solution and bacterial cells were allowed to percolate under gravitational flow. The obtained data from surface strengths and CaCO3 contents analysis of the consolidates soil specimens exhibited similar outcome ranging between 11448.00 ± 69.00 to 4826.00 ± 00 kPa and 5.56 ± 1.15 to 33.24 ± 0.59%, respectively. The data also indicated that replacing the costly cementation reagents with the technical-grade reagents resulted in 47 to 51-fold cost reduction. Finally, this thesis also investigated a feasible approach to scale-up the production of S. pasteurii cells using custom-built stirred tank reactor (3 m3) under the non-sterile condition from 216 L to 720 L and finally 2400 L. The non-sterile cultivation of the bacterial cells was intentionally designed to determine if the scale-up condition can support necessary urease activity and calcium carbonate precipitation needed to cement sand columns during in-situ treatment. After a total of 90 h incubation period, the bacterial cell cultures in 2400 L attained an OD of 1.8 ±0.09, pH of 9.2 ±0.05 and urease activity of 11.1 ±0.48 mM urea hydrolysed.min−1. The total calcium carbonate contents (19.0 ±1.58% to 39.4 ±1.88%) from the treated soil columns increased progressively with treatment cycles applied on different mould sets, while the difference of calcium carbonate contents between the top and bottom layers ranged from 3.8 to 7.2%. The findings of this thesis advocate that it is viable to scale-up the production of ureolytic bacterial cells and minimize bacterial cultivation cost. This was achieved by using technical-grade ingredients and custom-built reactor. The implications from this study suggest that future MICP field-scale applications which require a large concentration of ureolytic bacterial cultures can be performed under cost-effective conditions. iv “The universe does not give you what you ask for with your thoughts; it gives you what you demand with your actions” – Dr Steve Maraboli v DEDICATED TO MY DEAREST PARENTS (CLETUS AND MAGARET OMOREGIE) AND MY DARLING SISTERS (JENNIFER, SHARON AND THELMA) vi DECLARATION I, the candidate, declare that the contents of this thesis contain no material which has been previously accepted by me for the award of any other degree at any other university or equivalent institution. To the best of my knowledge, this thesis contains no material previously published or written by another person except where due references were made in the thesis. The contents of chapter 3, 4 and 5 have been published as research articles in peer-reviewed journals which are indexed by Clarivate Analytics (Science Citation Index) and Scopus. Relative contributions of all authors on the work that are based on published articles have been disclosed in the Authorship Indication Forms (see Appendix B). Also, I affirm that permission was obtained where necessary from the copyright owners to use third party copyright materials which were reproduced in this thesis (i.e. artwork, images or figures) or to use any of my published work (see Appendix A). Also, supplementary materials belonging to research articles for chapter 3, 4 and 5 are subsequently added at the end of each chapter sections. Armstrong Ighodalo Omoregie 19/09/2020 vii AUTHORSHIP INDICATION I would like to affirm the contributions of all co-authors of the articles which were included in this thesis as chapters (3, 4 and 5). The author indication forms for all published or submitted papers are provided in the appendix section of this thesis (see Appendix B). As shown in the authorship indication forms (see Appendix A), I confirm that major contributions (i.e. conceptualization, methodology, validation; data analysis, investigation; visualization and writing) of all papers included in this thesis were made by me, as indicated by my position as first author and corresponding author. viii ACKNOWLEDGEMENTS First and foremost, I wish to thank my coordinating supervisor, Assoc. Prof. Peter Morin Nissom, for the priceless support, motivation and prodigious suggestions he provided throughout my time at Swinburne University of Technology Sarawak campus (SUTS). I had the greatest opportunity to serve as a student under his supervision for both my MSc and PhD degree programs. You showed me how to properly communicate scientific data to audiences outside my research area which helped me obtain best oral and poster presenter at several l conferences held in Malaysia. Also, thanks for introducing me to durian. It has become one of my favourite Malaysian fruits. My supervisory team would not have been complete without Dr Dominic Ek Leong Ong, Prof. Enzo A. Palombo and Dr Tan Lee Tung. I am extremely thankfulyou’re your respective and incredible supports which came in form research grant provision, moral support and proofreading my manuscripts. My PhD research would not have been possible without the financial support (Swinburne Research Scholarship) from SUTS. This scholarship provided a full tuition waiver and monthly stipend for 3 years. Also, the School of Research (SoR) and Soletanche Bachy (Rueil-Malmaison, France) provided research grants that partly funded my PhD study. Also, I had the opportunity to secure discretionary funds that covered the expense of my conferences and laboratory analysis. I would also like to thank the (previous and present)