A Feasibility Study to Scale-Up the Production of Sporosarcina Pasteurii, Using Industrial-Grade Reagents, for Cost-Effective In-Situ Biocementation

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

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

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.

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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.

“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

DEDICATED TO MY DEAREST PARENTS (CLETUS AND MAGARET OMOREGIE) AND MY DARLING SISTERS (JENNIFER, SHARON AND THELMA)

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

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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.

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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) administrative staffs (specifically Jane PeiLing Teo and Dayang Salwa Binti Abang Nawaw) from the SoR for their tireless supports given to me and every postgraduate research candidate at SUTS. Also, I am tremendously grateful to the SUTS Laboratory Officers (Doris Liang, Nurul Arina Salleh, Cinderella Sio and Marclana Jane Richard) and the Biosafety Officer (Chua Jia Nia) from the school of Chemical Engineering and Science for their assistance during my time in the laboratories. You provided utmost assistance throughout my PhD experiment in the laboratories whenever needed. Jia Ni, I am so thankful for the biosafety guidelines and training especially relating to the outdoor bacterial scale-up production in the custom-built reactor. Thank you so much!

This three years PhD journey would not have been fulfilling without the support of a number of people whose contribution warrant acknowledgment and my sincere appreciation. First of all, special thanks go to Hasina Mohammed Mkwata whom I have known since 2011. I am so glad that you played a significant role in my life and will always be grateful for your remarkable supports. I am so glad we developed interest in science together, shared research ideas, collaborated and worked together under the supervision of Assoc. Prof. Peter M. Nissom. I wish you a very successful career and hope someday you become one of Tanzanian’s prestigious scientific researcher.

My heartfelt appreciation also to all my friends and colleagues namely, Stanley Nwobodo, Godswill Ejeohiolei Esechie, Darrell Nadeng Dominic, Chukwuka Christian Ohueri, Ghazaleh Khoshdelnezamiha, Nurnajwani Senian, AbdulKadir Muhammad Lawan, Dr Wallace Imoudu Enegbuma, Ts Dr Muhammad Khusairy Bin Bakri, Dr Augustine Chioma Affam and Dr Jibril Adewale Bamgbade. Thank you for generously finding time to participate in this PhD study which came in form of moral supports, friendship, ideas, suggestions and mentorship. It has been an astonishing pleasure to have known you all and be passionately influenced by you.

To Siti Naqiyah Seman, you came into my life, mid-way into my PhD journey at a critical time when I needed support. This PhD journey has been challenging, but you encouraged me throughout this academic endeavours and it ensured I stayed focused until the I reached the summit. I am also glad to have met your beautiful family. The hospitality, fondness and acceptance provided by your family has been a life changer. I also appreciate the moral supports and prayers rendered by your family, “Terima kasih”.

To my dearest parents and beloved siblings, thank you for loving and supporting me unconditionally since I left Nigeria in January 2007 to pursue my BSc, MSc and PhD degrees. It was never easy living outside the shores of Nigeria for over a decade without visiting home. I pray the future continues to bring sanctifying merriment to us, thanks again for the undying love and marvellous supports. I am so blessed to have a beautiful and kind- hearted family. Continuous thoughts about you all have been the inspirations which have propelled to me chase this academic success. May God bless you my dearest family!

TABLE OF CONTENTS

Abstract...... iii

Declaration ...... vii

Authorship Indication ...... viii

Acknowledgements ...... ix

List of Tables ...... xii

List of Figures...... xiii

List of Acronyms ...... xx

List of Appendices ...... xxii

Scholar Achievements ...... xxiii

Chapter 1: Introduction ...... 1

Chapter 2: Literature review ...... 27

Chapter 3: Cultivation of Sporosarcina pasteurii strain using food-grade yeast extract medium ...... 153

Chapter 4: Sand biocementation using Sporosarcina pasteurii strain and technical-grade cementation reagents ...... 166

Chapter 5: Scale-up production of Sporosarcina pasteurii cells using custom-built stainless steel stirred tank reactor ...... 189

Chapter 6: General conclusions and recommendations ...... 207

Appendix ...... 231

LIST OF TABLES

Table Title Page 2.1 List of ureolytic microorganisms isolated from various 39 indigenous locations. 2.2 Type of soils obtained from various locations treat with MICP 62 process 2.3 Previous large-scale and field-scale investigations on 66 biocementation for soil improvement 3.1 Nutrition contents of industrial-grade yeast extract 156 4.1 The two nutrient media used to cultivate S. pasteurii NB28(SUTS) 168 4.2 Cost analysis of the ingredients used in this study for bacterial 168 cultivation 4.3 Physiochemical properties of tap water used in this study 168 4.4 Growth kinetics of S. pasteurii NB28 (SUTS) in different 171 cultivation media 4.5 Comparison of technical-grade and analytical-grade elemental 174 compositions 5.1 Geometric parameters of the customized bioreactor tank 190 5.2 Experimental conditions for the sand mould sets 191 5.3 Growth kinetics of ureolytic bacteria when cultivated in the 192 custom-built reactor 5.4 DO, TDS and salinity of the bacterial seed culture samples 193 5.5 pH of effluents collected from the outlets of mould samples 193 6.1 Different bacterial species that are grown in the food-grade yeast 216 extract medium

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LIST OF FIGURES

Figures Title Page 1.1 A schematic diagram showing the production of cement clinker in a 3 rotary kiln. 1.2 The flow of research in this thesis. 14 2.1 Schematic representations of biominerals precipitation pathways 29 induced by biologically controlled mineralization (A), biologically induced mineralization (B) and biologically mediated mineralization (C) (Castro-Alonso et al., 2019). 2.2 Bar charts showing an increasing trend in the numbers of publications 33 on keywords (A) microbially induced carbonate precipitation and (B) microbial urease from 1999 to 2019. Source from Scopus on 14 January 2020 (keywords appearing in title, abstract and keywords: “microbially + induced + carbonate + precipitation, and “microbial + urease”). 2.3 Pie charts showing the categories of subject areas for research 34 documents published on keywords (A) microbially induced carbonate precipitation and (B) microbial urease from 1999 to 2019. Source from Scopus on 14 January 2020 (keywords appearing in title, abstract and keywords: “microbially + induced + carbonate + precipitation, and “microbial + urease”). 2.4 A schematic diagram showing the MICP process via ureolysis 35

pathway which eventually results in CaCO3 precipitation (Castro- Alonso et al., 2019).

2.5 Digital images showing microstructures of CaCO3 polymorphs 45 (Dhami et al., 2013). 2.6 Schematic diagram showing the effective bridge formation (Mujah et 61 al., 2017). Reprinted with permission from Taylor & Francis. 2.7 Schematic diagram showing different soil biocementation process 71 (Cheng & Cord-Ruwisch, 2012). Reprinted with permission from Elsevier.

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2.8 Results of a sandy soil sample subjected to MICP treatment via 72 surface percolation (Cheng & Cord-Ruwisch, 2014). Reprinted with permission from Taylor & Francis. 2.9 Chart showing the effect of different medium on urease activity of 73 enriched soil ureolytic bacteria (Cheng, Shahin, & Cord-Ruwisch, 2017). Reprinted with permission from Taylor & Francis.

2.10 Chart showing the UCS and CaCO3 content of treated soil done with 73 in-situ grown ureolytic bacteria (Cheng, Shahin, & Cord-Ruwisch, 2017). Reprinted with permission from Taylor & Francis. 2.11 Schematic diagram showing tank specimen subjected to injection 75 treatment method (Gomez et al., 2016). Reprinted with permission from the American Society of Civil Engineers. 2.12 Biocemented sandy soil observed after MICP treatment using the 75 injection technique (van Paassen et al., 2010). Reprinted with permission from the American Society of Civil Engineers. 2.13 Photos of bioslurry premixed with soil column before MICP 77 treatment (Cheng & Shahin, 2016) Reprinted with permission from Canadian Science Publishing. 2.14 Biocemented sand specimens subjected to MICIP premixing 78 treatment under different conditions; (A) sand without premix, (B) sand premixed with bacterial culture and (C) sand premixed with cementation reagents (Omoregie et al., 2018). 2.15 Surface strength of the treated sand samples from different treatment 79 groups after surface percolation treatment. Group 1 refers to sand columns which did not contain bacterial cells or cementation solution premix prior to treatment; Group 2 refers to sand columns which were premixed with only bacterial culture prior to treatment; and Group 3 refers to sand columns which were premixed with only

cementation reagents (urea and CaCl2) prior to treatment (Omoregie et al., 2018). 2.16 Schematic drawing of the batch reactor (A) and image of samples 81 after MICP submerged treatment method (B) (Zhao et al., 2014a).

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Reprinted with permission from the American Society of Civil Engineers. 2.17 Slope stabilization model test: (a) Schematic diagram of the 91 experimental arrangement of slope model and (B) profile of the cemented slope model (Gowthaman et al., 2019). Reprinted with permission from Springer Nature. 2.18 Arsenic concentration in soil fractions for the control and 94 bioremediated arsenic contaminated soil samples (Achal et al., 2012). Reprinted with permission from Elsevier. 2.19 Digital images showing repairs of cracks in shotcrete specimens 97 using MICP process after 28 days of curing period (Kalhori & Bagherpour, 2017). Reprinted with permission from Elsevier. 2.20 Instrumentation, MICP biogrouting and compression loading setup 101 (Lin et al., 2016). Reprinted with permission from the American Society of Civil Engineers. 2.21 Digital image showing pile specimen subjected to MICP treatment 102 for ground Improvement of soil (Lin et al., 2016). Reprinted with permission from the American Society of Civil Engineers. 2.22 Treated dunes with bacterial cells and cementation reagent for 104 penetration resistance experiment (Maleki et al., 2016). Reprinted with permission from Springer Nature. 2.23 PCB-contaminated concrete cylindrical specimens subjected to MICP 108 treatment (Okwadha & Li, 2011. Reprinted with permission from Elsevier. 3.1 The growth profile (A) and pH profile (B) of S. pasteurii 156 NB28(SUTS) cultivated in LG-YE (20 g.L-1) and FG-YE (20 g.L-1-). 3.2 Influence of the low-cost media concentration with urea (A) and 157 without urea (B) on biomass production. Vertical error bars indicate the means± SE of three independent repetitions. The alphabets indicate statistically significances (P < 0.05) as determined by one- way ANOVA with means comparison using Tukey-Kramer post hoc test.

3.3 Influence of the low-cost media concentration with and without urea 157 on urease activity. The difference between means sharing a common letter is not statistically significant at P < 0.05 as determined by one- way ANOVA with means comparison using the Tukey-Kramer post hoc test. The vertical error bars represent mean ±SD (n=3).

3.4 Influence of various initial pH medium on the biomass production 158 (A); pH (B) and urease activity (C) for S. pasteurii NB28(SUTS). The difference between means sharing a common letter is not statistically significant at P < 0.05 as determined by one-way ANOVA with means comparison using the Tukey-Kramer post hoc test. The vertical error bars represent mean ±SD (n=3). 3.5 Comparison of different selected laboratory-grade growth media with 158 the food-grade yeast extract media for their respective performances on growth (A); pH (B) and urease activity (C). The difference between means sharing a common letter is not statistically significant at P < 0.05 as determined by one-wayANOVA with means comparison using the Tukey-Kramer post hoc test. The vertical error bars represent mean ±SD (n=3)

3.6 The measurement of calcium carbonate contents (A) and pH of 159 effluents (B). The biocalcification test was performed with cementation solutions containing cementation solutions supplemented with different growth media and were incubated at 32oC for 24 h incubation without shaking. The difference between means sharing a common letter is not statistically significant at P < 0.05 as determined by one-way ANOVA with means comparison using the Tukey-Kramer post hoc test. The vertical error bars represent mean ±SD (n=3). 3.7 X-ray diffraction analysis of four selected precipitated samples 160 obtained after ureolysis process. Sample 1 (TSB); Sample 2 (AG- YE); Sample 3 (FG-YE) and Sample 4 (NB).

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4.1 The two cementation solutions containing technical-grade (A) and 169 analytical-grade (B) reagents before being inoculated with S. pasteurii. 4.2 Setup of sand columns prior to biocement treatment. 169 4.3 Monitored (A) growth and (B) pH profiles of S. pasteurii strain when 170 cultivated in Growth Medium-1 (GM-1) and Growth Medium-2 (GM-2). The vertical error bars represent mean ±SD (n=3).

4.4 Ureolysis-driven CaCO3 formation in beakers containing (A) 171 technical-grade cementation solution and solution (B) analytical- grade cementation solution after addition of S. pasteurii cultures.

4.5 XRD pattern of CaCO3 polymorphs obtained from (A) analytical- 172 grade and (B) technical-grade samples. 4.6 pH of the effluents collected from treated sand columns. The vertical 173 error bars represent mean ±SD (n=3). 4.7 ED-XRF spectrum results of the commercially available technical- 174 grade reagents used for biocement treatment. (A) yeast extract, (B) calcium chloride and (C) urea. 4. 8 Surface strength measurement of the biocemented sand samples using 174 pocket penetrometer. Treatments were carried out using ureolytic bacterial cultures with analytical-grade and technical-grade solutions. The acronyms AG (DW), AG (TW), TG (DW) and TG (TW) represent analytical-grade deionized water, analytical-grade tap water, technical-grade deionized water and technical-grade tap water, respectively. To determine whether the differences between group means are statistically significant at P < 0.05, one-way ANOVA with Tukey-Kramer post hoc analyses were used. The rectangular bars that share a letter (i.e. a, b, c, d, e, f) indicates there are no statistical significant differences between their means. The vertical error bars represent mean ±SD (n=3).

4.9 CaCO3 contents of the biocemented sand samples. Treatments were 175 carried out using ureolytic bacterial cultures with analytical-grade and technical-grade solutions. The acronyms AG (DW), AG (TW),

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TG (DW) and TG (TW) represent analytical-grade deionized water, analytical-grade tap water, technical-grade deionized water and technical-grade tap water, respectively. To determine whether the differences between group means are statistically significant at P < 0.05, one-way ANOVA with Tukey-Kramer post hoc analyses were used. The rectangular bars that share a letter (i.e. a, b, c, d, e, f) indicates there are no statistical significant differences between their means. The vertical error bars represent mean ±SD (n=3).

4.10 SEM analysis showing crystal morphologies of CaCO3 on surfaces of 176 the sand samples treated with S. pasteurii and 1.0 M of different cementation solutions. Analytical-grade (A) and technical-grade (B) reagents in deionized water; analytical-grade (C) and technical-grade (D) reagents in tap water.

4.11 SEM analysis showing the surface topography of CaCO3 after soil 177 biocementation was performed with S. pasteurii and 1.0 M of different cementation solutions. Analytical-grade (A) and technical- grade (B) reagents in deionized water; analytical-grade (C) and technical-grade (D) reagents in tap water. 5.1 Digital image of the custom-built stirred tank reactor (3 m3) used for 190 large-scale production of ureolytic bacteria. 5.2 An inner view of the custom-built reactor during scale-up process of 192 the bacterial cells. Sequentially, volume of cultures was increased from 216 L (A) to 2400 L (B). 5.3 Growth profile (A); pH profile (B) and urease activity (C) analyses 192 for ureolytic bacterial cultivation. The vertical error bars represent mean ±SD (n = 9).

5.4 CaCO3 content of different layers (top, middle and bottom) for the 193 biocemented sand samples. One-way ANOVA with Tukey-Kramer post hoc analyses were used to determine statistical significant (P < 0.05). The rectangular bars that share same letter indicates there are no statistical significant differences between their means. The vertical error bars represent mean ±SD (n=9).

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5.11 SEM micrographs showing different morphological crystal 193 formations for Mould 1 (A); Mould 2 (B) and Mould (C) after successful MICP treatment (3, 6 and 9 days respectively). 6.1 Digital image of biocemented sand specimens after treatment (6 212 months. 6.2 Digital image of biocemented sand specimens subjected to prolong 213 outdoor environmental conditions. Samples after 12 months (A) and 24 months of biocementation treatment.

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LIST OF ACRONYMS

Greenhouse gas GHG Ordinary Portland cement OPC Microbially induced carbonate precipitation MICP Enzyme-induced calcium carbonate precipitation EICP Microbial biopolymer formation MBF Water absorption test WAT Rapid migration test RMT Rapid chloride penetration test RCPT Microbial enhanced oil recovery MEOR Enhancing oil recovery EOR Removal of polychlorinated biphenyls PCBs X-ray diffraction XRD Food-grade yeast extract FG-YE Analysis of variance ANOVA Extracellular polymeric substance EPS Laboratory-grade yeast extract LG-YE Optical density OD Nutrient broth NB Tryptic soy broth TSB Luria broth LUB Fluid thioglycollate medium FTM Cooked meat medium CMM Lactose broth LB Marine broth MB Growth medium-1 GM-1 Growth medium-2 GM-2 Analytical-grade deionized water AG (DW) Analytical-grade tap water AG (TW) Technical-grade deionized water TG (DW) Technical-grade tap water TG (TW) Energy dispersive X-ray fluorescence ED-XRF

Scanning electron microscope SEM Energy dispersive X-ray EDX Tryptone soya agar TSA Ultraviolet UV

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LIST OF APPENDICES

Appendix Page Permission from the copyright owner 220 Authorship indication forms 231

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SCHOLAR ACHIEVEMENTS

Note: Names of the corresponding authors in the published papers are indicated by *

Journal Article Publications 1. Omoregie, A.I.*, Palombo, E., Ong, & Nissom, P.M. (2020), “Bioprecipitation of Calcium Carbonate Mediated by Ureolysis: A Review”, Environmental Engineering Research. [h-Index (ScimagoJR): 17; CiteScore (Scopus): 2.1; Impact Factor (©Clarivate Analytics): 1.087]. [manuscript No.: EER-D-20-00379] [current status: under review]. 2. Omoregie, A.I.*, Palombo, E., Ong, D.E.L., & Nissom, P.M. (2020), “A feasible scale-up production of Sporosarcina pasteurii using custom-built stirred tank reactor for in-situ soil biocementation”, Biocatalysis and Agricultural Biotechnology. 24, 101544. https://doi.org/10.1016/j.bcab.2020.101544 [h-Index (ScimagoJR): 30; CiteScore (Scopus): 2.8; Impact Factor (©Clarivate Analytics): Not available]. 3. Omoregie, A.I.*, Palombo, E., Ong, D.E.L., & Nissom, P.M. (2019), “Biocementation of sand by Sporosarcina pasteurii strain and technical-grade cementation reagents through surface percolation treatment method”, Construction and Building Materials. 228C, 116828. https://doi.org/10.1016/j.conbuildmat.2019.116828 [h-Index (ScimagoJR): 147; CiteScore (Scopus): 7.4; Impact Factor (©Clarivate Analytics): 4.419]. 4. Omoregie, A.I.*, Hei, N.L., Ong, D.E.L. & Nissom, P.M. (2019), “Low-cost cultivation of Sporosarcina pasteurii strain in food-grade yeast extract medium for microbially induced carbonate precipitation (MICP) application”, Biocatalysis and Agricultural Biotechnology, 17, 247-255. https://doi.org/10.1016/j.bcab.2018.11.030 [h-Index (ScimagoJR): 30; CiteScore (Scopus): 2.8 Impact Factor (©Clarivate Analytics): Not available]. 5. Omoregie, A.I.*, Ong, D.E.L. & Nissom, P.M. (2019), “Assessing ureolytic bacteria with calcifying abilities isolated from limestone caves for Biocalcification”, Letters in Applied Microbiology. https://doi.org/10.1111/lam.13103 [h-Index (ScimagoJR): 104; CiteScore (Scopus): 1.93; Impact Factor (©Clarivate Analytics): 3.5].

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6. Mkwata, H.M., Omoregie, A.I.*, Musa, I.B., Suyuh, J., Yee, P.H., Sing, L.W., Tan, L.T. & Nissom, P.M.* (2018), “A Laboratory Practicum on Screening for Lytic Bacteriophages from Soil Samples”, Transactions on Science and Technology, 5(4), 233-238. [h-Index (ScimagoJR): Not available; CiteScore (Scopus): Not available; Impact Factor (©Clarivate Analytics): Not available]. 7. Omoregie, A.I.*, Ginjom, I.R.H., & Nissom, P.M. (2018), “Microbial induced carbonate precipitation via ureolysis process: A mini-review”, Transactions on Science and Technology, 5(4), 245-256. [h-Index (ScimagoJR): Not available; CiteScore (Scopus): Not available; Impact Factor (©Clarivate Analytics): Not available]. 8. Omoregie, A.I.*, Siah, J., Pei, B.C.S., Yie, S.P.J., Weissmann, L.S., Enn, T.G., Rafi, R., Zoe, T.H.Y., Mkwata, H.M., Sio, C.A. & Nissom, P.M.* (2018), “Integrating Biotechnology into Geotechnical Engineering: A Laboratory Exercise”, Transactions on Science and Technology, 5(2), 76-87. [h-Index (ScimagoJR): Not available; CiteScore (Scopus): Not available; Impact Factor (©Clarivate Analytics): Not available]. 9. Omoregie, A.I.*, Khoshdelnezamiha, G., Senian, N., Ong, D.E.L., & Nissom, P.M. (2017), “Experimental optimisation of various cultural conditions on urease activity for isolated Sporosarcina pasteurii strains and evaluation of their biocement potentials”, Ecological Engineering, 109, 65-75. https://doi.org/10.1016/j.ecoleng.2017.09.012 [h-Index (ScimagoJR): 118; CiteScore (Scopus): 7.5; Impact Factor (©Clarivate Analytics): 3.512]. 10. Omoregie, A.I.*, Khoshdelnezamiha, G., Ong, D.E.L., & Nissom, P.M. (2017), “Microbial-induced carbonate precipitation using a sustainable treatment technique”, International Journal of Service Management and Sustainability, 2(1), 17- 31. https://doi.org/10.24191/ijsms.v2i1.6045 [h-Index (ScimagoJR): Not available; CiteScore (Scopus): Not available; Impact Factor (©Clarivate Analytics): Not available].

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Conference Presentations

1. “Biocementation: Using microbes to produce sandstones”, Innovation Technology Expo 24-25 July 2019, Imperial Hotel, Kuching, Malaysia. (Poster presentation). 2. “Large-scale Cultivation of Sporosarcina pasteurii Strain in a Low-Tech Customized Bioreactor for In-Situ Biocement Application”, 8th International Forum on Industrial Bioprocessing, 1-5 May 2019, Imperial Hotel, Miri, Malaysia. (Poster presentation). 3. “A study on the utilization of Sporosarcina pasteurii strain for low-cost biocement production in construction industry”, International Conference on Beneficial Microbes, 30 July-01 August 2018, The Waterfront Hotel, Kuching, Malaysia. (Oral presentation). 4. “Cultivation of Sporosarcina pasteurii NB28 (SUTS) Using Low-Cost Alternative Growth Medium for Biocement Application”, 3rd ASEAN Microbial Biotechnology Conference, 24-26 April 2018, Pullman Hotel, Kuching, Malaysia. (Poster presentation). 5. “Integrating Biotechnology into Geotechnical Engineering: A Laboratory Exercise”, The 3rd International Conference on Science & Natural Resources, 1-2 April 2018, Grand Borneo Hotel, Kota Kinabalu, Malaysia. (Oral presentation).

Awards

1. Awarded third place (bronze) for the category of technology and engineering poster section for the topic ‘Biocementation: Using microbes to produce sandstones’, at the Innovation Technology Expo (InTEX 19), Imperial Hotel, Kuching Malaysia which was organized by Universiti Malaysia Sarawak (UNIMAS). (July 25, 2019). 2. Award second place (runner-up) for an oral presentation titled ‘Bio-cementation: A sustainable construction technology’ at the Three Minute Thesis (3MT®) oral competition for PhD candidate which was organized by School of Research, Swinburne University of Technology Sarawak Campus. (September 20, 2017).

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Ad-Hoc Activities

1. Peer-Reviewed manuscripts submitted to Construction and Building Materials [Impact factor: 4.046; H-index: 129]; Journal of Basic Microbiology [Impact factor: 1.76; H-index: 47]; Malaysian Journal of Microbiology [Impact factor: Not available; H-index: 13]; Journal of Pure and Applied Microbiology [Impact factor: Not available; H-index: 12]; Journal of Archaeological Science: Reports [Impact factor: Not available; H-index: 16]; Waste and Biomass Valorization [Impact factor: 2.358; H- index: 28]; Environmental Engineering Research [Impact factor: 1.087; H-index: 14]; International Journal of Microbiology [Impact factor: Not available; H-index: 36]; and SN Applied Sciences [Impact factor: Not available; H-index: Not available]. 2. Elected President for Postgraduate Research Society (PGRS) at Swinburne University of Technology Sarawak Campus. (February 2018-March 2019). 3. Sarawak Postgraduate Representative for the Higher Degree by Research Representative Committee organized by the Faculty of Science, Engineering and Technology, Swinburne University of Technology, Melbourne Campus. (July 2018- June 2019). 4. Sarawak Postgraduate Representative for the campus development working group: new research innovation centre by Swinburne University of Technology Sarawak Campus. (May-December 2019).

Magazine article Omoregie, A.I. & Senian, N. (2017), “Building Bio-cement from Bacteria”, Discover Magazine: The Green Issue. Patodia, T. (Ed.) pp 34-37. Swinburne Sarawak Sdn. Bhd.

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Chapter 1

Introduction

1.1. Motivation of the study Cement is an important adhesion substance used in the construction industry for various applications (i.e. reservoirs, pavements, roads, dams, tunnels, bridges, mortar and bricks). It is by mass the largest manufactured product on earth and the second most used substance in the world after water (Scrivener, John, & Gartner, 2018). The global production of cement has increased tremendously since 1990 with an annual growth of 0.8–1.2%, and a predicted consumption rate of 3.7–4.4 billion tonnes by 2050 (Andrew, 2018; Benhelal, Zahedi, Shamsaei, & Bahadori, 2013). Although cement utilization has led to urban development and global economic growth, its production has regrettably resulted in the release of large amounts of greenhouse gases (GHG) such as carbon dioxide (CO2) into the environment (Enegbuma et al., 2020; Imbabi, Carrigan, & McKenna, 2012). Yet, the construction industry relies vehemently on cement utilization building and structural purposes. Despite the negative impacts cement causes to the environment at all stages of its manufacturing process, it has been able to revolutionize the construction and building practice.

Cement production requires 20–40% of energy and extremely high temperatures (950– 1500°C) for the transformation of limestone to clinker, which is a key constituent of cement production (Ivanov, Chu, & Stabnikov, 2015). CO2 emissions occur during the calcination of limestone in a cylinder rotary kiln to produce clinkers (Fig. 1.1). The burning of fossil fuels (i.e. coal) is typically used to provide energy for heating of the raw materials, thus accounting for 40–50% of GHG emissions released into the environment (Van Den Heede & De Belie, 2012). The remaining 50–60% GHG emissions results from the decomposition of limestone upon heating. Ordinary Portland Cement (OPC) is the most versatile a type of cement used globally for a wide range of construction applications (i.e. reservoirs, pavements, roads, dams, tunnels, bridges, mortar and bricks) (Ivanov et al., 2015). Unfortunately, there is a demand for alternative ecological-friendly and sustainable building materials. It has been reported that the global GHG emissions increased by 2% in

2018, thus reaching 51.8 gigatonnes of CO2 emissions after six years of monitoring, with minimal annual growth of around 1.3% (Olivier & Peters, 2019).

Fig. 1.1: A schematic diagram showing the production of cement clinker in a rotary kiln.

The unfortunate increase in global GHG emission was partly attributed to the increase of global CO2 emissions from fossil consumption by industrials including cement industry. Also, recent global emissions statistics showed that China, European Union, India and the United States of America are the top emitters of GHG, while China singularly emits more than 26% of the global GHG emissions (United Nations Environment Programme, 2019). Environmental pollution associated with increased cement production is due to the rapid growth of human populations and increased demand for cement. Thus, it is imperative that solutions are developed which reduce the undesirable effects of producing cement materials for construction (Enegbuma et al., 2020). The push for a reduction in global GHG emissions is supported by governments around the world because they understand that neglecting this issue will cause serious and detrimental effects to humans and the environment (Imbabi et al., 2012). This concern led to ‘The Paris Agreement’ by member countries of the United Nations Framework Convention on Climate Change to tackle climate change through intensified actions and investments required for a sustainable low carbon future (Falkner, 2016).

Also, societal concern for more ecological-friendly industrial production has increased the scrutiny of manufacturing processes and measures in the commercial sector (Mirkouei, Haapala, Sessions, & Murthy, 2017). An enormous increase in the detrimental hazards to the environment from construction and building activities has also spun the sensitisation among construction stakeholders around the world (Enegbuma et al., 2020). Hence, major cement producers now promise to use of zero or minimal GHG emission technologies for their operations by 2050 (United Nations Environment Programme, 2019). Driven by environmental responsibility and concerns, new innovative studies are being initiated to improve current construction materials and develop resilient sustainable building materials (Ehrenfeld, 2008; Manzini, 2009). Modern construction now desires a built environment which utilizes less cement-based materials (Scrivener et al., 2018). Cement is often used more than needed for several construction applications where fillers and other cementitious materials can serve as alternatives that can significantly minimize the production of GHG emissions during the making of building materials (John, Damineli, Quattrone, & Pileggi, 2018; Shanks et al., 2019).

Researchers are now working on innovative technologies that could help reduce environmental and public health issues caused due to cement production. This need has shifted the focus into using sustainable materials and processes. Green building construction is being developed as an alternative strategy to reduce energy consumption and the overall impact of the built environment on our natural environment (Ohueri, Enegbuma, & Kenley, 2018). The GHG emission and air pollutants from cement production can be reduced through the use of other technologies and strategies (Enegbuma et al., 2020). The construction industry could play an imminent role in improving “green construction” practices and products which are critical to solving many energy and sustainability-related problems (Duraisamy, 2016). It requires the utilization and implementation of ecological-friendly substitutes of conventional cement materials.

1.2. Background of the study The rapid urbanization and industrialization due to increase in human population have led to demand for more cementitious materials that has unintentionally played a substantial role in environmental pollution and health problems. Henceforth, the need to resolve this issue has resulted in tremendous researches which also involves the use of living microorganisms. This led to the introduction of Construction Biotechnology which has emerged as a new sub-disciple of Microbial Biotechnology that uses microorganisms and their products (i.e. enzymes and biominerals) for development of construction materials.

Over the past 20 years, civil engineers and biotechnologists have fervently studied the use of microorganisms for the improvement of soil mechanical properties through microbially induced carbonate precipitation (MICP) pathways. The MICP has resulted in the development new microbial materials (biocementation and construction processes (biocementation). Although, biocement can serve as a replacement to conventional materials (i.e. Portland cement) due to its low viscosity when required to cement soils with smaller pores or fill cracks, it however should not be seen as a new alternative to conventional cement. Biocementation technique is used to produce inorganic minerals (i.e.

CaCO3) which serves as a cementitious agent to enhance the strength of granular soil and reduce its permeability. However, the production of ureolytic bacteria for in-situ biocementation represent a major cost factor that hinders its industrial potential (Cheng, 2012). Biocementation utilizes ambient temperature and less energy (10%) for the manufacturing of conventional cement (Ivanov et al., 2015; Myhr et al., 2019). Biocementation does not require pyro-processing method for production of cementitious building materials, instead it uses microbial catalysts to induce cementing materials. Recently, there is noticeable increase in global patent filings for the use of biocementation technology from multidisciplinary researchers to produce sustainable cementitious materials (Dapurkar & Telang, 2017). However, despite many successful studies were conducted under laboratory-condition, the use of this technology for field-scale implementation to help reduce global GHG footprint is still at its infancy (Myhr et al., 2019).

Since early human civilization, microorganisms have been used for production of industrially-relevant enzymes and molecules vital for numerous applications (Steele & Stowers, 1991). These beneficial microorganisms are either utilized in their natural or mutated forms. Utilization of these beneficial microorganisms dates back to early 6000 BC when the Babylonians and Sumerians used yeast to produce alcoholic beverage from barley (Singh, Kumar, Mittal, & Mehta, 2016). Enzymes are biomacromolecules that accelerate chemical reactions by converting substrates into different molecules (products) and have been widely implementation commercially for chemical production, biofuel production, food processing and ingredients, beverage manufacturing, agricultural and feed usage, degradation of textile, leather and polymer, medical and biopharmaceutical usage (Kumar & Murthy, 2016; Kunduru, Kutcherlapati, Arunbabu, & Jana, 2017; Singh et al., 2016). Microbial enzymes are also comparatively more stable and diverse than enzymes derived from plants and animals (Alves et al., 2014). Enzymes from microbial sources are preferred due to their availability in large quantities and low production cost. The physiological and physico-chemical properties of microbial enzymes are easy to regulate (Ibrahim, 2008). Besides, the microorganisms can be easily manipulated to obtain desired enzymes having targeted characteristics (Kar, Husaini, & Tasnim, 2017).

In 2014, the global market value for microbial enzymes was estimated around USD 4.2 billion with a compound annual growth rate of approximately 7 %, and it is expected to reach approximately USD 6.2 billion in 2020 (Singh et al., 2016). It was also suggested that in 2021, the enzyme market for technical applications and process development will be more successful in the Asia-Pacific and North America regions (Chapman, Ismail, & Dinu, 2018). Without any doubt, microorganisms and their enzymes are highly efficient for human usage and have economic value. Studies on microorganism and their beneficial enzymes have become ubiquitous. For example, glucoamylase, amylase, cellulase, lactase, are among the prominent enzymes which are produced from microorganisms such as Marasmius cladophyllus, Clostridium thermocellum, Aspergillus niger and Bacillus amyloliquefaciens (Khan & Husaini, 2006; Kumar & Murthy, 2016; Rezaul Kar et al., 2017). List of various microorganisms, their corresponding enzymes and industrial applications was tabulated in a research paper by Singh et al., (2016).

Enzyme urease (urea amidohydrolase; EC 3.5.1.5) is another well-known enzyme that is accentuated in this study and has also gained huge attention in recent decades from cross- disciplinary researchers. Urease is capable of hydrolysing urea and connected with protein degradation (Mobley, Island, & Hausinger, 1995). Urease was initially considered to be a potent virulence factor in pathogenic bacteria (i.e. Helicobacter pylori, Proteus mirabilis, Campylobacter pyloridis, and Staphylococcus saprophyticus) (Kumari et al., 2016). Until recently, urease was mostly used as biosensors to quantify or detect urea in blood and urine samples (Francis, Lewis, & Lim, 2002). It has a growing demand in fertilizer and food industries to serve as a reducing agent for the preparation of alcoholic beverages and urease inhibitors for urea hydrolysis rate reduction.

Sporosarcina pasteurii is a highly active ureolytic microorganism which has been widely studied for its potential applications in the construction industry. Although, urease production occurs in numerous bacterial species, yet, several studies have shown that ureolytic activity is often associated with pathogenic bacteria (Konieczna et al., 2013). However, S. pasteurii is known to be the most appropriate candidate for biomineralization activity due to its high urease production and versatility (Oualha, Bibi, Sulaiman, & Zouari, 2020). Enzyme production by this microbe has been well documented in the literature (Der Star et al., 2009; Omoregie et al., 2017; van Paassen, 2009; Williams, Kirisits, & Ferron, 2016). Sporosarcina pasteurii is mostly known for its causative ability to induce sufficient

CaCO3 precipitates through MICP occurrence (Ghosh, Bhaduri, Montemagno, & Kumar, 2019). Based on German Technical Rules for Biological Agents on the classification of prokaryotes (bacteria and archaea), S. pasteurii is a risk group 1 microorganism, making it unlikely to cause human disease (Ivanov, Stabnikov, Stabnikova, & Kawasaki, 2019). This might be the reason most researchers prefer using S. pasteurii for construction and building purposes because it is non-pathogenic. Conventional MICP studies showed that S. pasteurii aided fluid permeability reduction and sand consolidation (Ferris & Stehmeier, 1992; Kantzas et al., 1992). This discovery motivated other researchers to carry out more studies on S. pasteurii for biotechnological and engineering applications. One of its most successful applications to date is on ground improvement or soil solidification. Further comprehensive studies and utilization of MICP technology are discussed in more details in Chapter 2 section of this thesis (Literature Review).

Development of biomediated geotechnical process through S. pasteurii for soil stabilizations have focused on identifying, controlling and applying biogeochemical process, optimizing and modifying abiotic factors for chemical reactions and urease production, modifying abiotic chemical reactions, using active ureolytic cells and process to improve CaCO3 precipitates within soil matrix (Burbank et al., 2013; DeJong & Kavazanjian, 2019; DeJong, Mortensen, Martinez, & Nelson, 2010; Omoregie et al., 2017). The growth of extracellular polymeric substance (EPS), a gel-like secretion substance generated by ureolytic bacterial cells has been shown to coat granular soil particles and improve their mechanical performance (Cunningham, Characklis, Abedeen, & Crawford, 1991). New studies on the importance of microbial enzymology and their kinetic associations which are required for industrial microbial purpose are essential (Khan & Husaini, 2006). Their relation to various parameters especially in fermenters for large-scale production of cells for their beneficial enzymes should also be investigated. The research reported in this current thesis discusses the possibility of reducing the cultivation cost of ureolytic bacterial cells and scale-up feasibility using custom-built reactor. Also, it discusses in-vivo and in-situ soil biocementation using low-grade reagents as replacement to high-grade reagents.

1.3. Problem statement Most MICP studies are performed using laboratory-grade or analytical-grade reagents (Achal, Mukherjee, & Reddy, 2010; Dhami, Reddy, & Mukherjee, 2016; Li, Zhang, Ge, & Yang, 2018). Some of these media (i.e. yeast extract, luria broth, nutrient broth) are habitually used to supply essential nutrients for the cultivation of various ureolytic microorganisms (Achal & Pan, 2014; Cheng & Cord-Ruwisch, 2013; Thiyagarajan et al., 2016). The cultivation medium constitutes ingredients which contain the macro-nutrients and micro-nutrients required to provide or maintain cellular biosynthesis and metabolism. They are also useful in regulating protein structures and functions of enzymes. Appropriate cultivation medium and substrate are among the basic environmental necessity that is crucial for the cultivation of microbial cells.

Fortunately, most ureolytic bacterial species used for MICP experiment whether procured from culture collection centres (i.e. American Type Culture Collection; National collection of industrial and marine bacteria; German Collection of Microorganisms and Cell Cultures; and China General Microbiological Culture Collection Center) (Harkes et al., 2010; Kim & Youn, 2016; Okwadha & Li, 2011; Omoregie et al., 2016; Raut, Sarode, & Lele, 2014; Sidik, Canakci, Kilic, & Celik, 2014) or obtained from the natural environments (i.e. soil, cave, sewage and water) (Al-Thawadi & Cord-Ruwisch, 2012; Burbank, Weaver, Williams, & Crawford, 2012; García, Márquez, & Moreno, 2016; Omoregie, Ong, & Nissom, 2019) can be easily grown with any microbiological media either broth-based or agar-based that constitute enough ingredients to support microbial cell development.

They do not often require special formulations or extremely high density (i.e. nutritionally rich) of growth medium for sufficient microbial cell formation. The development of commercial media which are used as a general-purpose medium used for cultivation for a wide variety of microorganisms typically contain ingredients such as peptone, beef extract, casein enzymic hydrolysate, dextrose (glucose), tryptone and sodium chloride. These compositions provide essential amino acids, inorganic salts, carbohydrate, vitamins, minerals and sometimes growth factors, buffering agents or hormones to the microbial cells. The varying concentrations of these aforementioned components can provide energy, nutrients, essential metals or mineral required to sustain proliferation and maintenance of microbial cells. Since these commercially manufactured growth media are of usually high-grade or high-purity, unfortunately, they are rather too cost-prohibitive for any MICP field-scale trials. Similar to cultivation media, analytical-grade chemicals (i.e. urea, calcium salts) are critical for CaCO3 precipitation during MICP experiments. Urea substrate is either included (to stimulate urease production) or excluded in cultivation medium for bacterial growth. For biocement experiments, varying concentrations of urea and calcium chlorides, depending on the design of experiments, are often prepared together as constituents of cementation solution to induce appropriate CaCO3 contents.

In certain cases, other chemicals (i.e. ammonium chloride, sodium sulphate, sodium chloride, nickel, magnesium chloride, iron (II) sulphate are also utilized for enrichment culture, cultivation medium and to stimulate biominerals precipitation (Bansal, Dhami, Mukherjee, & Reddy, 2016; Burbank, Weaver, Green, Williams, & Crawford, 2011; Omoregie et al., 2019; Osinubi et al., 2019; Wang, Jonkers, Boon, & De Belie, 2017). Irrespective of the brands or purity-level procured from the manufacturer for MICP experiments, they are rather suitable for small-scale MICP experiments but too costly for large-scale trials. The use of laboratory-grade or high-purity reagents for MICP experiments would drive up the production cost especially for the biotechnological methods. Not only that, but only limited studies in the literature also scale-up the cells of ureolytic bacteria for numerous MICP studies (Cheng & Cord-Ruwisch, 2013; Whiffin, 2004). The implication of this issue will result in limited MICP field-scale or large-scale experiments, expensive production of bacterial cells and MICP treatments. Also, knowledge about the effects of MICP parameters on the behavior of ureolytic bacterial cells in the scale-up process will be limited. Finally, the actualization of industrial or commercial MICP implementation will continue to be delayed or limited.

Interestingly, a few studies in the literature have shown that researchers are concerned about the cost of MICP process and have experimented on various ways to reduce the materials or reagents used for MICP experiments. The MICP process is dependent on both microbial and chemical activities for the production of urease and biominerals desired for cell growth and biocalcification process. Hence, substrate and cementation reagents that can facilitate low-cost scale-up of ureolytic bacterial cells and in-situ soil treatment were highly needed. The substitutes which have been utilized are eggshells, lactose mother liquor, waste stream, seawater, pig urine and pilot-scale custom-made reactor (Achal, Mukherjee, Basu, & Reddy, 2009; Arias, Cisternas, & Rivas, 2017; Chen et al., 2018; Choi, Wu, & Chu, 2016; Der Star et al., 2009; Whiffin, 2004). Results from these studies have been promising but there are certain drawbacks (i.e. usage complexity or availability) may limit their future selection for MICP studies. Therefore, MICP researchers must provide more solutions to the high cost of MICP process.

1.4. Thesis aim and specific objectives

The main aim of this study was to investigate the efficacy of using inexpensive commercially available technical-grade reagents and custom-built reactor to scale-up the production cells of Sporosarcina pasteurii for in-situ soil biocementation.

The specific objectives set out to achieve the research aim of this thesis are: i. To determine the feasibility of cultivating S. pasteurii strain in an economical food-grade yeast extract medium and investigate its effect on bacterial production, urease activity and biocalcification; ii. To evaluate the feasibility of using commercially available and inexpensive technical-grade reagents as replacements to the costly analytical-grade reagents for bacterial cultivation and soil biocalcification; and iii. To investigate the feasibility to scale-up the production of S. pasteurii from small- scale to large-scale using a custom-built stainless steel stirred tank reactor (3 m3) under non-sterile conditions and determine its suitability for soil biocementation.

1.5. Scope of the study The scope of the study in this thesis was conducted and presented from an applied biotechnology perspective. Throughout this study, Sporosarcina pasteurii NB28 (SUTS) was the ureolytic bacterial strain used previously isolated by Omoregie et al., ( 2017) from Sarawak limestone cave sample. The study focused on investigating the possibility to scale- up the cells production of ureolytic bacteria for soil biocementation from viewpoints of cost reduction (Fig. 1.2). The studies reported in Chapter 3 and Chapter 4 were done under laboratory-conditions in Swinburne University of Technology Sarawak Campus and with small-scale experimental design. On the other hand, Chapter 5 was carried out outdoor but inside the campus after obtaining approval from the Biosafety Committee.

The studies presented in this thesis were designed to demonstrate that microbial cell production and MICP soil treatments could be performed inexpensively. Therefore, series of techno-economic analysis were carried out based on scientific experiments which involved using microbial catalyst (S. pasteurii), laboratory propagation and production of S. pasteurii using food-grade yeast extract for biocementation, using technical-grade cementation chemicals to reduce the cost of microbial catalysts and MICP treatment, and scale-up of ureolytic bacterial cells to 2400 L using custom-built reactor under non-sterile condition. The outcome of the results was compared to cultivation and soil treatment done with the costly or high-grade reagents to provides the suggestions that technical-grade reagents can lower the bacterial cultivation and soil biocementation treatment costs from an economic viewpoint. It may also significantly lower the cost of scaling up the production from the perspective of cultivation medium.

To achieve this goal, the scope of this thesis was divided into three phases; The first phase of the thesis utilized a biotechnological approach to investigate the likelihood of replacing laboratory-grade cultivation medium with a cheap substrate. Food-grade yeast extract (Angel Yeast Co. Ltd) media which is commercially available in bulk quantity was procured and compared with eight selected microbiological-based media. The cheap substrate contained mostly yeast extract (50%) and maltodextrin (40%), hence, to validate the suitability for bacterial cultivation, biomass, urease activity and biomineralization performances were determined.

The second phase of this thesis utilized microbiological and geotechnical engineering skills which included microbial cell cultivation and optimization of culture condition, determination of pH level, urease activity and CaCO3 contents, scale-up of bacterial cells in the custom-built reactor, soil percolation and treatment, and compressive strength of treated soil columns. As to the extent of which level the MICP cost may be lowered from treatment perspective which requires biotechnological and geotechnical engineering implementation, laboratory-based MICP test on small scale soil columns was carried out. The study investigated the effectiveness of performing soil biocementation using inexpensive technical-grade cementation reagents as a replacement to the costly analytical- grade cementation reagents.

Both the low-grade and high-grade reagents were prepared in both deionized water and tap water at different concentration (0.25-1.0 M) and used for soil treatment which was carried out triplicates to ensure repeatability. The influence on urea hydrolysis, surface strength, CaCO3 contents, microstructural structures of soils were evaluated. The performance of the bacterial cells when cultivated in low-grade and high-grade cultivation both prepared in tap water and deionized water were studied before soil biocementation.

The third phase of this thesis investigated the sequential scale-up production of ureolytic bacterial cells under the non-sterile condition to lower down the cost of MICP process, promote and ease the setting up and implementation of the in-situ biocementation at the construction sites. This evaluation incorporated the reagents (media and chemicals) previously reported to lower MICP cost. Hence, the study focused on the feasibility to scale-up the cells of S. pasteurii using a custom-built stainless steel tank reactor (3 m3) under non-sterile and in-situ conditions. The non-sterile cultivation condition did not hinder propagation of microbial cells nor affect urease activity needed to induce CaCO3 precipitation for in-situ MICP implementation. Technical-grade reagents were also used in phase 3 study for bacterial cultivation and in-situ soil biocementation. In the custom-built reactor, the bacterial cells were sequentially scale-up from small scale production (216 L) to pilot-scale production (720 L) and subsequently large-scale production (2400 L). The growth, pH, urease activity of the cells, when cultivated in the custom-built reactor, were evaluated. Furthermore, the pH effluents from the sand moulds after MICP treatment were measured, microstructural characterization and calcite distribution in the treated soil specimens after the curing period were also evaluated.

Cultivation of Sporosarcina pasteurii NB28(SUTS) Soil biocementation using Sporosarcina pasteurii and Scale-up production of Sporosarcina pasteurii using using food-grade yeast extract medium technical-grade cementation reagents custom-built stainless steel stirred tank reactor

Fig. 1.2: The flow of research in this thesis.

1.6. Significance of the study

This research investigated ways and solutions to further minimize the costing of large- scale bacterial cells production for in-situ biocementation that can be applicable at the industrial sites. The work displayed in this thesis has the potential to solve numerous biotechnological and engineering problems especially if it requires site treatment and cell cultivation. Since sterility is not of high-priority as long urease production and biocalcification are not lowered or inhibited, the bacterial scale-up process in a budgeted custom-built stirred tank reactor (3 m3) is of great advantage. This is because it will encourage more future MICP studies to move away from the conventional laboratory-based experiments to in-situ pilot-scale, large-scale and field-scale experiments. The use of procurable inexpensive technical-grade reagents and the custom-built reactor will be able to lower future MICP cultivation and treatment costs to a minimal level. In addition, the outcome of this thesis would pave the way for a new frontier with a wide range of possible biotechnological applications which require the usage of a technical-grade substrate and custom-built reactor for large-scale microbial production.

This research provides a vehement knowledge on how low-cost cultivation of ureolytic bacterial cells for biocementation of soil can be performed. Series of investigation done showed the feasibility replacing high-purity (analytical-grade) reagents with low-purity (technical-grade) reagents for bacterial cultivation and also biocalcification. The major contribution arising from this study is in the use of non-sterile reactor to scale-up the cells of ureolytic bacteria through a sequential technique. This is the first MICP investigation on large-scale production of ureolytic bacterial cells using a custom-built reactor. Typically, to attain a required volume of seed culture (3 m3), several reactors in greater order will be required to scale-up the bacterial cells and would increase the cultivation cost. Consequently, this thesis showed that with a custom-built reactor, the bacterial cells were successfully propagated to a final volume of 2400 L. The entire cultivation process was done in-situ and under the non-sterile condition to mimic industrial situation. More so, application of the ureolytic bacterial cells and technical-grade cementation reagents for

MICP treatment on soil column showed that the urease activity and CaCO3 precipitation was feasible despite cultivating the S. pasteurii in a non-sterile reactor.

1.7. Thesis outline This thesis presented herein consist of six chapters: Introduction (Chapter 1); Literature review (Chapter 2); Cultivation of Sporosarcina pasteurii strain using food- grade yeast extract medium (Chapter 3); Sand biocementation using Sporosarcina pasteurii strain and technical-grade cementation reagent (Chapter 4); Scale-up production of Sporosarcina pasteurii cells using custom-built stainless steel stirred tank reactor (Chapter 5); and General conclusions and recommendations (Chapter 6). Concluding remarks are provided at the end of each chapter (except for chapter 1) to highlight and summarise the key outcomes of each chapter. Brief description of each chapter discussed in this thesis is presented below.

Chapter 1 is the introductory chapter of this thesis. It starts by discussing the motivation behind this research; background of biocementation as a new alternative technology suitable for soil improvement and stabilization. The aim and specific objectives, scope of the study; the significance of the study; and thesis outline were also presented in this chapter.

State-of-the-art literature review on MICP technology and biocementation for soil stabilization are presented in Chapter 2. This chapter discusses the various pathways involved in MICP; urease enzyme; its mechanisms and CaCO3 polymorphisms. This was followed by a review on the factors affecting biocementation, different methods used for soil biocementation. The advantages and limitations of MICP technology were also included in this chapter.

The first-key research of this thesis is presented in Chapter 3 which was mainly focused on the viability of replacing costly laboratory-grade growth media with inexpensive food-grade yeast extract media for the cultivation of S. pasteurii. The chapter starts by comparing the growth and pH profile of S. pasteurii in high-grade and low-grade yeast extract medium. The performance of this low-cost cultivation medium was also compared with eight selected laboratory-grade growth medium. The obtained results in this chapter were presented on the basis of biomass, pH and urease activity, biomineralization test and identifying the precipitated CaCO3 polymorphs.

The second key- research of this thesis is presented in Chapter 4 which focused on investigating the feasibility of using commercially available inexpensive technical-grade cementation reagents as a suitable replacement to the costly analytical-grade cementation reagents mostly used for soil biocementation. Comparable tests were performed based on biomineralization test, XRD analysis, soil biocementation using different concentration (0.25–1.0 M) prepared in deionized water and tap water. Further analyses such as microstructure analysis, surface strength test, CaCO3 contents and pH effluent analysis used to evaluate the suitability of these technical-grade cementation reagents for field-scale implementation were presented in this chapter.

The third and final-key research of this thesis is presented in Chapter 3 which was mainly focused on examining the scalability of S. pasteurii from small-scale to large-scale by using a custom-built stainless steel stirred tank reactor (3 m3) under non-sterile condition. Using technical-grade cultivation ingredients, the scalability of ureolytic bacterial cells was carried out from 216 L to 720 L and finally 2400 L. The chapter showed that the biomass and urease activity of the bacterial cells were not hindered during economic scale-up process. In addition, in-situ soil biocementation in different sand moulds were carried out and the performance of the MICP was evaluated after treatment. The moulds were treated with different cycles (3 days, 6 days and 9 days) of cementation solution (1 M). Measurements of CaCO3 contents, microstructure analysis and pH effluent analysis were also presented in this chapter. The chapter was able to suggest that using a custom-built stirred tank reactor was able to scale-up the production of ureolytic bacterial cells under the non-sterile condition and was feasible for field-scale MICP implementation.

Lastly, Chapter 6 provides an overview of the findings (major contributions) obtained from the results and discussion sections of the thesis, along with the conclusion of the study. The summaries in this chapter focused on the outcome of the main investigation in this thesis (Chapter 3, Chapter 4 and Chapter 5) and are presented within the context of one another. Recommendation for future studies to be considered is also given in this chapter.

1.8. References Achal, V., Mukherjee, A., Basu, P. C., & Reddy, M. S. (2009). Lactose mother liquor as an alternative nutrient source for microbial concrete production by Sporosarcina pasteurii. Journal of Industrial Microbiology and Biotechnology, 36(3), 433–438. doi:10.1007/s10295-008-0514-7

Achal, Varenyam, Mukherjee, A., & Reddy, M. S. (2010). Biocalcification by Sporosarcina pasteurii using corn steep liquor as the nutrient source. Industrial Biotechnology, 6(3), 170–174. doi:10.1089/ind.2010.6.170

Achal, Varenyam, & Pan, X. (2014). Influence of calcium sources on microbially induced calcium carbonate precipitation by Bacillus sp. CR2. Applied Biochemistry and Biotechnology, 173(1), 307–317. doi:10.1007/s12010-014-0842-1

Al-Thawadi, S., & Cord-Ruwisch, R. (2012). Calcium carbonate crystals formation by ureolytic bacteria isolated from Australian soil and sludge. Journal of Advanced Science and Engineering Research, 2, 12–26.

Alves, P. D. D., Siqueira, F. de F., Facchin, S., Horta, C. C. R., Victória, J. M. N., & Kalapothakis, E. (2014). Survey of microbial enzymes in soil, water, and plant microenvironments. The Open Microbiology Journal, 8(1), 25–31. doi:10.2174/1874285801408010025

Andrew, R. M. (2018). Global CO2 emissions from cement production, 1928–2017. Earth System Science Data, 10(4), 2213–2239. doi:https://doi.org/10.5194/essd-11-1675- 2019

Arias, D., Cisternas, L. A., & Rivas, M. (2017). Biomineralization mediated by ureolytic bacteria applied to water treatment: A review. Crystals, 7(11). 345–359. doi:10.3390/cryst7110345

Bansal, R., Dhami, N. K., Mukherjee, A., & Reddy, M. S. (2016). Biocalcification by halophilic bacteria for remediation of concrete structures in marine environment. Journal of Industrial Microbiology and Biotechnology. 43, 1497–1505. doi:10.1007/s10295-016-1835-6

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Chapter 2

Literature review

2.1. Introduction Biomineralization is the biochemical process where inorganic mineral deposition occurs within or outside the cells of microorganisms (Boskey, 2003). The resulting products are composites of mineral and organic constituents (Osinubi et al., 2020). In all cases of biomineralization, microbial cells control the formation of biominerals through the expression of proteins (acting as nucleators) or enzymes which modify the functions of these proteins, or by regulating the ionic transport system (Boskey, 2003). Microbial synthesis of biominerals as shown in Fig. 2.1 occurs via active or passive processes consisting of:

(i) biologically controlled mineralization (development of conditions such as growth, morphology and composition that are regulated by specific cellular activity in the microorganism) (Phillips et al., 2013); (ii) biologically mediated mineralization (biominerals are passively precipitated due to the presence of cell surface organic matter such as extracellular polymeric substances (EPS) linked to biofilms without the need of extracellular or intracellular biological activity) (Castro-Alonso et al., 2019; Dhami et al., 2013); and (iii) biologically induced mineralization (metabolic activity of microorganisms alters the environment to favour the induction of crystal formation by acting as nucleation sites, thus indirect precipitation is due to interactions between by-product released by the microorganism and available ions in the environment) (De Muynck, De Belie, & Verstraete, 2010; Stocks-Fischer, Galinat, & Bang, 1999).

The reproducibility process of biominerals (i.e. carbonates, oxides, sulphide, silicates and phosphates) that occurs naturally are meticulously difficult to replicate unlike the conventional synthetic process (Mamo & Mattiasson, 2019). There are more than 60 biomineral groups that are formed by organisms and 50% when confirmed comprises of calcium-bearing minerals (Weiner & Dove, 2003). Like other biomineralization process, MICP occurs under active biological settings and favourable environments to generate desired CaCO3 mineral formation.

(A)

(B)

(C)

Fig. 2.1: Schematic representations of biominerals precipitation pathways induced by biologically controlled mineralization (A), biologically induced mineralization (B) and biologically mediated mineralization (C) (Castro-Alonso et al., 2019).

Currently, the acronym “MICP” is used in various publications to describe “microorganisms induce calcium carbonate precipitation”, “microbiologically-induced calcite precipitation”, “microbiological carbonate precipitation”, microbially induced calcite precipitation” and “microbial carbonate precipitation” (Anbu, Kang, Shin, & So, 2016; Arias, Cisternas, & Rivas, 2017a; Bachmeier, Williams, Warmington, & Bang, 2002; Mujah, Shahin, & Cheng, 2017; Zhu & Dittrich, 2016). However, early descriptions of the potential of MICP referred to the mechanism as “bacteriogenic precipitation of minerals”, “microbially mediated calcium carbonate precipitation”, “bacterially induced carbonate mineralization” and

“microbiological precipitation of CaCO3” (Ferris, Stehmeier, Kantzas, & Mourits, 1997; Rodriguez-Navarro, Rodriguez-Gallego, Chekroun, & Gonzalez-Muñoz, 2003; Stocks-Fischer et al., 1999; Warren et al., 2010). MICP is governed by six factors, namely the concentration of calcium ions, the concentration of dissolved inorganic carbon, pH level, genetics of urease genes, CaCO3 polymorphism and availability of nucleation sites (Hammes & Verstraete, 2002; Sarayu, Iyer, & Murthy, 2014). However, it has been recently shown that induced EPS is an

important factor which can influence MICP by providing nucleation sites to trigger CaCO3 formation (Kim et al., 2015).

2.2. Microbially Induced Carbonate Precipitation Calcium carbonate is one of the most abundant minerals on earth and comprises of 4% in weight of the earth’s crust (Castro-Alonso et al., 2019; Krajewska, 2018). The production of

CaCO3 minerals induced by microorganisms has attracted numerous researchers globally for various potential applications (Dhami, Reddy, & Mukherjee, 2013). Calcium carbonate precipitation is common in marine waters, sediment, freshwater, soil and other environments (Castro-Alonso et al., 2019). Calcium carbonate is widely available as a natural inorganic compound (i.e. limestone and marble, coral, shellfish and snail shell). Its availability and accessibility have made it one of the most versatile materials known. Both naturally obtainable

and precipitated CaCO3 are used globally for various industrial applications, e.g. sealant,

asphalt, pharmaceutical, food, paper and toiletries. The use of CaCO3 precipitated through MICP for soil stabilization has gained enormous interest among researchers.

The MICP process utilizes microbial activities to induce CaCO3 formation (Choi et al.,

2020). The use of CaCO3 biominerals precipitated through MICP process for granular soil stabilization has gained enormous interest among researchers. The MICP technology has many interesting applications that could solve ongoing environmental problems (Arias et al., 2017a). The understanding of soil behaviour for over three centuries has focused on mechanical principles, geological processes and mineralogy, however, research in biotechnology and earth science has allowed crucial contribution of microorganism which mediate minerals formation and geochemical reactions (Mitchell & Santamarina, 2005). There are two notable mechanisms namely, enzyme-induced calcium carbonate precipitation (EICP) and microbial biopolymer formation (MBF) that can be used as alternatives to MICP for biomineral precipitation, depending on the research purpose or application. MICP typically requires three components

(urease-secreting microorganism, calcium ion and urea) for CaCO3 precipitation to occur (Chae, Chung, & Nam, 2020). However, EICP is a rather simple and straightforward process but is relatively expensive because it requires procurement of urease. For EICP, commercial urease, which is available in powdered form, can be used instead of microbially sourced urease.

Similar to MICP, EICP induces CaCO3 formation through catalysis of urea, however urease is sourced from plants such as jack bean (Canavalia ensiformis), soya bean (Glycine max), pumpkin (Cucurbita maxima), watermelon (Citrullus vulgaris), potato (Solanum tuberosum) and pigeon pea (Cajanus cajan) (Dilrukshi & Kawasaki, 2019).

The EICP does not require complex cultivation of microbial cells, nutrient supply for survivability or oxygen availability which can make controlling enzymatic activity difficult (Abdullah, Hamed, & Edward, 2018). On the other hand, MBF requires a mixture of microbial cells and EPS for biofilm production (Choi et al., 2020). Microorganisms also use of MBF as a strategy to survive and adapt in adverse conditions or environments (Malheiro & Simões, 2017). Biofilm formations can protect microorganisms from external factors (i.e. physical, chemical and biological), with major effect on soil bonding, weathering of minerals, degradation of organic matters and biogas generation (Choi et al., 2020). The microbial cells attach to the surface for initial multiplication, maturation and production of the biominerals before detachment (Malheiro & Simões, 2017). While all three biomineralization processes (MICP, EICP, and MDF) are promising, MICP is the most popular process for numerous studies specifically involving soil solidification.

MICP has rather become the most successful and selected process which can easily induce

the vast amount of CaCO3 formation for numerous applications especially soil solidification.

Besides, The precipitation of CaCO3 via MICP process is governed by six factors (concentration of calcium ions, the concentration of dissolved inorganic carbon, pH level,

genetics of urease genes, CaCO3 polymorphism and availability of nucleation sites) (Hammes & Verstraete, 2002; Sarayu et al., 2014). The importance of MICP technology is reflected in the steady increase in the number of research publications describing laboratory-based experiments and field-based studies for real applications (Zhu & Dittrich, 2016). Fig. 2.2 shows that 410 and 3041 publications with keywords “microbially induced carbonate precipitation”, and “microbial urease”, respectively, were recorded in the Scopus database from 1999 to 2020. Although there are fewer articles on “microbially induced carbonate precipitation”, and “ureolytic” overall, there has been noteworthy increase since 2013.

It is vital to highlight that, while microbial urease was not popularly used for research or industrial purposes until recently, plant urease (i.e. from jack bean and soybean) was well known for its tremendous source of high enzymatic activity and there are available published papers which goes back to the 19th century. On the other hand, publications with the keyword “ureolytic” showed a steady increment, while “microbial urease” saw an exponential growth in the recorded publication from double- to triple-digit numbers. This suggests that the interest in the industrial applications in microbial urease is increasing. Fig. 2.3, clearly display that this is a multi-disciplinary research field that has attracted the interest of researchers from various expertise. The figure (Fig. 2.3) also suggested that most published work available in Scopus database for the selected years and keywords are in the subject area of Environmental Science (17%) and Agricultural and Biological Sciences (30%). On the other hand, the subject areas with the lowest publication records are in Energy (2%) and Chemical Engineering (2%).

90 (A) 80

400 (B) 350

300

250

200

150

100

Fig. 2.2: Bar charts showing an increasing trend in the numbers of publications on keywords (A) microbially induced carbonate precipitation and (B) microbial urease from 1999 to 2019. Source from Scopus on 14 January 2020 (keywords appearing in title, abstract and keywords: “microbially + induced + carbonate + precipitation, and “microbial + urease”).

Chemistry Energy (A) Chemical Engineering 4% 2% 4% Earth and Planetary Agricultural and Sciences Biological Sciences 23% 5%

Biochemistry, Genetics and Molecular Biology 8%

Materials Science Environmental 9% Science 17%

Immunology and Microbiology 11% Engineering 17%

Engineering Chemical Engineering 3% (B) Pharmacology, Chemistry 2% Taxicology and 3% Pharmaceutics Agricultural and 3% Biological Sciences 30% Earth and Planetary Sciences 5%

Biochemistry, Genetics and Molecular Biology 8%

Medicine 9%

Immunology and Environmental Microbiology Science 12% 25%

Fig. 2.3: Pie charts showing the categories of subject areas for research documents published on keywords (A) microbially induced carbonate precipitation and (B) microbial urease from 1999 to 2019. Source from Scopus on 14 January 2020 (keywords appearing in title, abstract and keywords: “microbially + induced + carbonate + precipitation, and “microbial + urease”).

2.3. MICP via ureolysis pathway

There are six known MICP pathways that result in in the saturation of CaCO3 crystals, namely, urea hydrolysis (ureolysis), photosynthesis, dissimilatory sulphate reduction, denitrification (nitrate reduction), ammonification of amino acids and methane oxidation (Anbu et al., 2016; Castanier, Le Métayer-Levrel, & Perthuisot, 1999; Zhu & Dittrich, 2016).

However, ureolysis is the least complex and most widely preferred CaCO3 precipitation process for numerous applications. The ureolysis pathway is also straightforward and results in 90% chemical conversion efficiency of calcite within a short duration (Mujah et al., 2017). More so, the sulphate reduction pathway is another MICP process which is widely investigated for its biominerals capacity, unfortunately, it does not get sufficient attention for engineering application (Zhu & Dittrich, 2016). Ureolysis-driven MICP process is determined by the breakdown of urease which results in CaCO3 precipitation. During microbial urease activity (Fig. 2.4), urea hydrolysis occurs to form equimolar amounts of ammonia and carbonic acid. It has been recently shown that, in support of urease, carbonic anhydrase converts the carbonic acid to bicarbonate and ammonia hydrolysis results in the production of two moles of ammonium and hydroxide ions (Castro-Alonso et al., 2019). These products equilibrate in the solution before the pH of the microenvironment is affected (Dhami et al., 2013).

Fig. 2.4: A schematic diagram showing the MICP process via ureolysis pathway which eventually results in CaCO3 precipitation (Castro-Alonso et al., 2019).

Irrespective of the pH level of the medium, urea hydrolysis leads to increased alkalinity and in the presence of added soluble calcium ion sources, calcium carbonate precipitation occurs. This is also because the intracellular accumulation of calcium ions leads to excessive expulsion of protons, thus making the cells to export calcium to compensate for the loss of protons (Castro-Alonso et al., 2019). This then leads to supersaturation of carbonate precipitates induced on the surfaces of the cells. Sufficient supersaturation state of the calcium ions favours the growth of microbial cells by reducing the toxic calcium production in its microenvironment (Sarayu et al., 2014). This suggests that the mechanism of carbonate precipitation is used to reduce the high calcium ion concentrations but the chloride ions which 2+ will be present together with Ca ions when added as CaCl2, may be toxic much before the calcium ions have any impact on the cells. The key role of microorganisms has been attributed to its ability to produce urease and allow enhanced local pH level (Dhami et al., 2013; Mitchell

and Ferris, 2005). Also, the bacterial cell surface is critical for CaCO3 precipitation because of the presence of a negatively charged group, thus allowing positively charged metal ions (i.e. Ca2+) to bound with the bacterial cell surface (Castanier et al., 1999; Rivadeneyra, Delgado, Ramos-Cormenzana, & Delgado, 1998). This occurrence favours heterogeneous nucleation site which leads to the production of carbonate crystals deposition.

2.4. Microbial urease Urease represents an historically important milestone because it was the first enzyme to be crystallized and shown to contain nickel (Weeks, Eskandari, Scott, & Sachs, 2000). The scientific interest on microbial urease was largely due to its enzymatic activity because gastritis and stomach cancer are associated with infection from Helicobacter pylori (Mobley, Garner, & Bauerfeind, 1995). Also, the role of microbial urease in enzymatic activity was primarily linked to the recycling of nitrogenous wastes and nitrogen assimilation (Mora & Arioli, 2014). Urease is produced by various taxonomically diverse microbial species, including normal flora and non-pathogens (Fisher, Yarwood, & James, 2017; Sarayu et al., 2014). Microbial enzymes are regarded to be comparatively more stable and diverse than enzymes derived from plants and animals (Alves et al., 2014).

Microbial enzymes can be easily controlled physiologically and physio-chemically, are produced in large have quantities and can be inexpensively extracted using downstream processes (Binod et al., 2013; Ibrahim, 2008). Bacterial ureases are different to those from other sources (i.e. plant and fungal ureases) because they are composed of the heteromeric subunit (Krajewska, 2018). Irrespective of the source and structural composition, urease contains a nickel centre with a characteristic structure (Krajewska, 2018). It has been suggested that urease-producing microorganisms can generate adenosine triphosphate through ureolysis and ammonium production (Al-Thawadi, 2011; Mobley & Hausinger, 1989). Similar to other bacterial regulatory systems, urease enzyme genes are organized in operons gene clusters (Castro-Alonso et al., 2019; Sarayu et al., 2014). Urease genes (ureABIEFGH) are all transcribed in the same direction, which ae vital for an active site synthesis (Mobley et al., 1995). Accessory genes (ureD, ureE, ureF and ureG) encode the accessory activation proteins which synthesize the active urease to enhance assembly of the nickel-dependent site of the enzyme (Sarayu et al., 2014) Some bacterial species (e.g. H. pylori) contain two subunit genes (ureA and ureB), while other such as Bacillus sp. contain more subunit genes (Castro-Alonso et al., 2019). The urease produced by Sporosarcina pasteurii consists of three subunits with two nickel atoms in the active site (Benini et al., 1999). This bacterium produce intracellular urease close to 1% dry weight of cells (Bachmeier et al., 2002). Therefore, it is imperative to

stress that more investigations should focus on how the subunit genes of urease induce CaCO3 precipitation. Understanding how their environmental factors may influence these genes is vital.

In the search for bacterial species able to precipitate carbonate minerals, many investigators have focused on screening soils. Microorganisms constitute between 70–85% of all living components within soil and it is estimated that a single kilogram of soil contains between 109 and 1012 microbes at the surface (Benini et al., 1999; Mitchell & Santamarina, 2005). Many researchers have reported the isolation of diverse microbial species from various soils capable of producing urease and CaCO3 precipitates (Table 2.1). Results from these investigations showed that the types of bacteria used for MICP experiments affects the enzyme activity, crystal formation and, ultimately, the overall outcome of the cementation process. S. pasteurii is a highly active ureolytic bacterium which has widely been studied for its potential applications in the construction industry. Although urease production occurs in numerous bacterial species, ureolytic activity is often associated with pathogenic bacteria (Konieczna et al., 2013).

S. pasteurii is non-pathogenic and is considered the most appropriate candidate for biomineralization activity due to its high urease production and versatility (Oualha, Bibi, Sulaiman, & Zouari, 2020). Indeed, enzyme production by S. pasteurii has been well documented in the literature (Der Star et al., 2009; Omoregie et al., 2017; van Paassen, 2009;

Williams, Kirisits, & Ferron, 2016) and this microbe is known for its ability to induce CaCO3 precipitates through MICP (Ghosh, Bhaduri, Montemagno, & Kumar, 2019). Based on the German Technical Rules for Biological Agents on the classification of prokaryotes (bacteria and archaea), S. pasteurii is a classified as a Risk Group 1 microorganism, making it unlikely to cause human disease (Ivanov, Stabnikov, Stabnikova, & Kawasaki, 2019). This might be the reason most researchers prefer using S. pasteurii for construction and building purposes. Some of the earliest uses of S. pasteurii involved novel fluid permeability reduction and sand

consolidation through precipitation of CaCO3 (Ivanov, Stabnikov, Stabnikova, & Kawasaki, 2019; Kantzas et al., 1992).

Table 2.1: List of ureolytic microorganisms isolated from various indigenous locations Microorganisms Isolation sites Purpose of studies Summary of findings Authors

Penicillium Cement sludge Determined the The calcification medium showed increased pH Fang et al., chrysogenum from China. efficiency of fungal (up to 10.5) and urease activity (42.5 U.L-1), (2018) CS1 mycelia to act as bio- while the compressive strength of bio- based fiber in sandstone formed was 1800 kPa. cementation.

S. pasteurii Cave samples Investigated the effect of The experimental results showed that urease Omoregie strains from Sarawak, various parameters on activities were optimum at 25 to 30°C, pH 6.5 et al., Malaysia. urease activity for four to 8.0, 24 h incubation and 6 to 8% (w/v) urea (2017) locally isolated ureolytic concentration, while the surface compressive bacteria strength ranged from 573.33 to 700 psi.

Aspergillus niger Unspecified Explored the optimum Optimal enzyme conditions for the fungi strains Khan et al., strains sample source, conditions for maximum 6.0 to 10.0 g (biomass), 3.0 to 5.0 mL (2019) Gujrat, Pakistan. urease enzyme (inoculum size), 1.5 to 2.0 g (nitrogen), 150 to production from wheat 250% (moisture content), 7.0 to 9.0 (pH), 35oC straw under the solid- (temperature) and 0.88 to 2.63 U.L-1 (urease state fermentation activities). process.

Bacillus Sewage sample Reported a novel The optimum conditions for biomineralization Helmi et licheniformis from approach to produce were YE-UR-Ca growth medium (20 g.L−1 of al., (2016) −1 −1 Governorate, CaCO3 under optimized yeast, 20 g.L of urea and 50 g.L of pure Egypt. conditions for calcium), initial pH medium of 8 to 10, bioconsolidation of incubation temperature of 30 to 35°C and urea mortar layers. concentration resulting to urease enzyme of 10 mg.mL−1.

Enterobacter Several samples Focused on the isolation Maximum urea degrading capacity were Ghashghaei ludwigii (soil, freshwater, of calcite forming between 125 to 2883 NH4+.L-1.h-1, pH and chalk, cement, bacteria from natural behaviour were between 7.0 to 9.0, and Emtiazi,

and activated environments for microscopic observation of CaCO3 crystals (2013) sludge) from biotechnological sizes ranged from 35 to 63 nm. Isfahan, Iran. applications.

Arthrobacter Concrete and Focused on isolating and Morphological analysis of the crystals was Montaño- crystallopoietes, mortar samples characterizing native between 45 to 243 μm, showed different crystal Salazar et Rhodococcus from Bogotá, ureolytic bacteria for structures (rhombohedral, fibro-radial al., (2018) qingshengii and Colombia. MICP applications. spherulite and spherical calcite) and the Psychrobacillus compressive strength of treated specimen was psychrodurans 11.6% high than the control specimen.

Bacillus Calcareous soil Investigated the isolation The calcite precipitates, maximum urease Dhami et megaterium, samples from and characterization of activities, carbonic anhydrase production and al., (2013) Bacillus cereus, Anantapur calcifying bacterial observed extracellular polymeric substances Bacillus District, Andhra strains and determine produced were 187 mg.100 mL-1, 690 U.mL-1, thuringiensis, Pradesh, India. their abilities to produce 115 U.mL-1 and 36.4 nmol.mL-1 for B. Bacillus subtilis, urease and biominerals megaterium; 167 mg.100 mL-1, 620 U.mL-1, 85 and Lysinibacillus precipitates. U.mL-1 and 33.5 nmol.mL-1 for B. fusiformis thuringiensis; 178 mg.100 mL-1, 587 U.mL-1, 90 U.mL-1 and 26.4 nmol.mL-1 for B. cereus; 152 mg.100 mL-1, 525 U.mL-1,70 U.mL-1 and 28.6 nmol.mL-1 for L. fusiformis and; 156 mg.100 mL-1,515 U.mL-1, 60 U.mL-1 and 26.8 nmol.mL-1 for B. subtilis, respectively.

Sporosarcina sp., Fluid samples Studied the presence of After the 13 days cultivation, out of the 57 Graddy et Bacillus sp. and from the bio- ureolytic diversity at the isolates, 47 strains were ureolytic bacteria al., (2018) Oceanobacillus stimulation tank, end of bio-stimulated belonging to the genera of Sporosarcina sp., sp. California, United MICP experiment. Bacillus sp. and Oceanobacillus sp. States of America.

Pararhodobacter Soil sample near Evaluated the whole cells It was reported that urease activity of the cell Fujita et al., sp. strain SO1. beachrock in urease activity of culture increased with increase in biomass (2017) Okinawa, Japan. Pararhodobacter sp. growth, while maximum urease activity of SO1, and effect on Pararhodobacter sp. were obtained at 60°C various conditions (i.e. and pH 8. In addition, The sodium dodecyl temperature and pH). sulfate polyacrylamide gel electrophoresis of soluble fraction for the ureolytic bacteril cell showed were between 15 to 60 kDa.

Bacillus cereus Yangtze River MICP process was Bacillus cereus YR5 was able to grow on the Kumari et YR5 sample near employed by for Cr(VI) acetate minimal medium (250 mg.L-1 of Cr(VI) al., (2014) Chongming reduction from soil. and after the bioremediation test, exchangeable County, Cr(VI) was reduced from 100 to 1.86 mg.kg-1 Shanghai, China. when compared to the control sample 39.3 mg.kg-1.

Bacillus cereus Peat soil from Focused on screening, Preliminary results showed that the isolates had Phang et strains Sarawak, isolating and their optimum biomass and urease activity at al., (2018) Malaysia. characterizing the urease pH 9 except for one strain (pH 4), also they

activities of ureolytic could all induce CaCO3 precipitation. bacteria from acidic tropical peat specimens.

Sporosarcina Nursery garden Performed a preliminary The isolates had high removal rates (88% to Li et al., globispora soil from Beijing, evaluation of ureolytic 99%) of heavy metals (nickel, copper, lead, (2013) (UR53), China. microorganisms for zinc, cadmium and cobalt) after 48 h of Sporosarcina heavy metal removal by incubation. Also, the microstructural analysis koreensis (UR47), precipitation. showed different crystal formations Sporosarcina sp. (rhombohedral, sphere and needle-shaped) R-31323 (UR31) when the pH medium was between 8 to 9. and Bacillus lentus (UR41).

Sphingobacterium Metal mine sites The objectives of this Out of the 93 isolated ureolytic bacterial Kang et al., sp., Enterobacter in Gangwondo, study were to isolate and isolates, only 6 were selected due to high (2015) cloacae and Korea characterize Pb-resistant urease activity and calcite precipitation. Lysinibacillus ureolytic bacteria and to Besides, two of these selected isolates were sphaericus. utilize the urease activity able to successfully reduce lead heavy metal of these strains for Pb concentration from 5.9 mg.L-1 and 7.2 mg.L-1 removal through to 2.3 to 2.7 mg.L-1. biomineralization

Sporosarcina Calcareous rock Aimed at synthesizing The measured ammonia concentration from the Kumar et pasteurii strain samples from the in-vitro calcareous treated sand column were 187.30 ±66.73 mM al., (2019) Tamil Nadu and 8.32 mM a, respectively. Also, result from

coastal area, sand-rock through MICP the compressive strength test ranged from 1000 India. process. to 3100 kPa.

Bacillus Soil specimen The preliminary study The ureolytic bacterial strain that distinctly Mekonnen paramycoides, from Addis aimed at characterizing different substrate utilization profiles, whereas et al., Citrobacter Ababa, Ethiopia. the carbon substrate there was strong metabolic similarity d between (2019) cedlakii and utilization patterns of the E. bugadensis and C. sedlakii. Some substrates Enterobacter microbial isolates. (i.e. Glucose, Maltose and Fructose) were used bugadensis. by the isolates but were unable to mediate some other substrate (i.e. Tween-40, D-Arabitol, D- Lactic Acid, Methyl Ester, D-Fucose, Quinic Acid, and D-Aspartic Acid).

Bacillus sp. Samples (soil and To perform a The specific growth rate (0.09.h-1), maximum Stabnikov VUK5, water) from comparative study on urease activities (6.2 and 8.8 mM hydrolysed et al., Bacillus sp. VS1 Singapore, Kiev, isolated ureolytic urea.min-1) and compressive strength test (765 (2013) and Ukraine and the bacteria from different and 845 kPa) for Bacillus sp. VS1 and VUK5 Staphylococcus Dead Sea in climate zones. strains were similar. However, Staphylococcus succinus. Jordan, succinus was not selected due to its hemolytic respectively. and toxigenic capability.

Microbial activities influence the production of CaCO3 biominerals which can result in different polymorphs of CaCO3 crystals as shown in Fig. 2.5. There is a growing number of

the available information on the cause of CaCO3 polymorphism which occurs during biomineralization (Sarayu et al., 2014). Currently, the polymorphs of CaCO3 exist in the anhydrous form (calcite, aragonite and vaterite), hydrated crystalline form (monohydrocalcite and ikaite) and several amorphous phases with a diverse degree of hydration ranges (Chen et al., 2009; Dhami et al., 2013; Hammes et al., 2003a; Xu et al., 2006). It has been suggested that

CaCO3 crystal morphology are specific to different bacterial strains and amino acid sequences (Hammes et al., 2003). More so, vaterite and calcite are regarded as the most commonly

precipitated polymorphs of CaCO3 by bacteria (Rodriguez-Navarro et al., 2012).

Fig. 2.5: Digital images showing microstructures of CaCO3 polymorphs (Dhami et al., 2013).

2.5. Factors influencing MICP proficiency The MICP requires biochemical and microbial activities in the physical environment to occur which can influence the outcome of this process. A large number of biotic (i.e. microbial genotype and cell concentration) and abiotic (i.e. nutrient and chemical concentrations) factors are responsible for the success of MICP (Wong, 2015) Successful MICP relies on sufficient

urease production and CaCO3 precipitation, both of which can interchangeably affect soil cementation. For successful field-scale implementation, the aforementioned factors should be comprehensively studied and their optimized. During field-scale trials, other factors will be unavoidable influences on MICP treatment (Mortensen et al., 2011). Although there are several additional factors that can impact MICP such as temperature (Mortensen et al., 2011; van Paassen, 2009); initial relative densities (Al Qabany, Soga, & Santamarina, 2012; Khan et al., 2019); curing duration (Zhao et al., 2014b); reaction time (Khan et al., 2019; Zhao et al., 2014a); varying soil particle sizes (Al Qabany et al., 2012; Wong, 2015); organic matter content (Oliveira & Neves, 2019); oil contaminants and freeze-thaw cycle ( Cheng, Shahin, & Mujah, 2017); treatment cycle and flow (Cheng, Shahin, & Mujah, 2017; Nayanthara, Dassanayake, Nakashima, & Kawasaki, 2019); and anoxic condition (Martin et al., 2012). However, only the four major contributors will be discussed in this review

2.5.1. Bacterial genotype and cell concentration Of the many different microorganisms that have been widely reported to produce urease and cause CaCO3 precipitation, members of the genus Bacillus are the most common ureolytic bacteria isolated from local sources. However, S. pasteurii from the Sporosarcina group is the most used microorganism for multiple MICP applications (Anbu et al., 2016). The selection of appropriate microorganisms for specific MICP applications is critical because different bacterial genotypes can result in diverse MICP outcomes. Some bacterial species may be able to generate greater biomass in a short period or require a less enriched nutrient medium for cultivation, however, they may not be suitable for MICP due to their toxicity, acidity and

insufficient production of urease. The mechanism required to induce CaCO3 crystal formation may also be affected if the bacteria are unable to efficiently increase the pH of the microenvironment (Mamo & Mattiasson, 2019).

The nature of exopolysaccharide secretion by different microorganism can also result in different MICP treatment process, thus it is essential to determine the appropriate ureolytic bacteria to use for various MICP applications (Mamo & Mattiasson, 2019). For example, B. megaterium, Pararhodobactor sp., Lysinibacillus sphaericus and S. pasteurii are often used for the improvement of soil solidification, heavy metal remediation and crack repair especially in moderate to high temperate climate regions (30–60°C) (Achal, Pan, Zhang, & Fu, 2012; Fujita et al., 2017; Omoregie et al., 2017; Soon, Lee, Khun, & Ling, 2014). Conversely, Lysinibacillus xylanilyticus has recently been reported to be a suitable MICP agent for soil improvement in cold regions (i.e. 15–25°C) (Gowthaman et al., 2019). The nature of exopolysaccharide secretion by different microorganism would also result in different MICP treatment process, thus it is essential to determine the appropriate ureolytic bacteria to use for various MICP applications (Mamo & Mattiasson, 2019).

The concentration of bacterial cells used for MICP is another factor which needs consideration. The amount of bacterial cells used per unit volume for MICP influences the level of biomineral formation (Mamo & Mattiasson, 2019). Ureolytic activity is dependent on the available substrate (e.g. urea) and the concentration of biomass cells, hence determining how the bacterial cell concentration influences urease production should be determined (Mortensen et al., 2011). Imran et al., (2018) recently reported that the number and diameter of crystal particles showed increase with higher bacterial (Pararhodobacter sp) cell concentration in their experiment. The cementation reagents and the bacterial cell concentration are two primary

factors which control the degree of CaCO3 content homogeneity with respect to strength increment over the depth of the treated biocemented soil column (Nayanthara et al., 2019). It has been suggested that higher bacterial cell concentrations (106–108 cells.mL-1) increases the amount of CaCO3 content during MICP (Okwadha & Li, 2010). This is because the cells are used to provide nucleation sites necessary to initiate the creation of an alkaline environment to

induce growth of CaCO3 precipitates (Stocks-Fischer et al., 1999). Okwadha and Li (Okwadha 2- & Li, 2010) showed that the increase in bacterial cells resulted in more CO3 production in the

cementation solution, but this is not entirely supported by other studies. A higher rate of CaCO3 precipitation during MICP does not result in homogenous biocalcification of tested soil specimens (Mamo & Mattiasson, 2019).

Zhao et al., (2014a) also investigated how various cell concentrations influences soil biocementation. They cultivated S. pasteurii until OD 0.3–1.5 and they observed that the urease

activity, compressive strength, and CaCO3 content were all significantly influenced by an increase in bacterial cell concentration. In another study, which investigated the effect of bacterial cell concentration (OD 0.5–1.5) on the physico-mechanical property of cement mortar, it was reported that OD 1.0 resulted in highest strength and lowest water absorption values (Abo-El-Enein, Ali, Talkhan, & Abdel-Gawwad, 2013).

2.5.2. Cementation reagents and its concentrations The presence of cementation reagents such as urea and Ca2+ ions are essential for the ureolysis to occur. Firstly, ureolytic bacteria utilize urea as a source of nitrogen and energy, thereby allows the enzymatic hydrolysis of urea by urease for the production of ammonia and carbonate ions (Anbu et al., 2016; Mobley & Hausinger, 1989; Stocks-Fischer et al., 1999). Since ureolysis pathway permits alkaline microenvironment around the cell to occur by raising the pH (i.e. pH 9) due to the release of ammonia gas, sufficient urea must be used to calibrate the discretion of excess alkaline environment that may hinder optimum ureolytic activity and bacterial necessary for optimum MICP process (Al-Salloum et al., 2017; De Muynck et al., 2010). Also, the concentration of cementation reagents must be controlled to achieve evenly

distributed CaCO3 precipitation because if this is not optimized, precipitation which may occur can result in excess crystal formations that can lead to bioclogging (Mortensen et al., 2011).

Based on the mechanism of bacterial consolidation to induce CaCO3 crystals, urea and calcium sources are the basic substances used in bacterial bonding (Tang et al., 2017). Since biocalcification may rely more on Ca2+ than urea concentration which may be attributed to the fact that the added Ca2+ source is not metabolically utilized, though gets accumulated outside

the bacterial cells for more precipitation of CaCO3 crystals (Hammes et al., 2003; Mamo & Mattiasson, 2019; Okwadha & Li, 2010). From the chemical perspective of MICP process, the concentration of carbonate and calcium sources are extremely important for the determination

of CaCO3 precipitation (Mamo & Mattiasson, 2019). For this biominerals to occur, an 2+ 2- appropriate amount of Ca (supplemented externally for mixture) and urea (for CO3 production) need to be provided (Al-Salloum et al., 2017).

The CaCl2 is commonly used by researchers as a precursor for producing CaCO3 crystals. However, unfortunately, chloride ions are regarded as harmful to building and construction materials because they can lead to corrosion or degradation of the pores structures. Using the Building Research Establishment Digest 264 as a practice guideline for chloride categorization, chloride ion with concentration below 0.4% by mass of cement, between 0.4% to 1.0% by mass of cement, and above 1% by mass of cement, has a low, medium and high risk level, respectively (Goyal, Pouya, Ganjian, & Claisse, 2018). Determining the corrosion and defects of chloride ion concentration can also be presented in ratio of chloride ion to hydroxide ion (Alonso, Andrade, Castellote, & Castro, 2000; Figueira, Sadovski, Melo, & Pereira, 2017). Higher chloride binding for total chloride contents often lead to higher chloride threshold ratio which can vary between 0.3 to 40.0 (Bamforth, 2004). Hence the appropriate amount of CaCl2 should be used during MICP treatment experiments which often ranges between molar ratio of

1.0 to 1.5, while for CaCO3 crystallization to occur during ureolysis, the molar ratio of ions to urea ranges between 0.5 to 2.0 (Ivanov & Stabnikov, 2017, 2019). Some researchers have suggested the use of other calcium sources for MICP process which have different outcome due to the kinetics of biochemical reactions (Al-Salloum, et al., 2017). Although, when compared with other calcium sources, it is widely suggested by researchers that calcium chloride is the most common used calcium for soil biocalcification (Tang et al., 2017).

The alternative calcium sources which have been successfully used to induce CaCO3

crystals are calcium acetate [Ca(C2H3O2)2], calcium oxide [CaO], calcium nitrate [Ca(NO₃)₂],

calcium formate [Ca(HCOO)2], calcium lactate [C6H10CaO6] and calcium diglutamate [Ca(C₅H₈NO₄)₂] (Achal & Pan, 2014; De Muynck, Cox, Belie, & Verstraete, 2008; Fahmi et al., 2018; Jing & Wu, 2014; Mamo & Mattiasson, 2019; Ronholm et al., 2014; Tayebani & Mostofinejad, 2019). Reports from these aforementioned alternative calcium sources have been

promising outcomes and may be used to replace CaCl2 if required. It has been suggested that

the use of alternative calcium source such as Ca(C2H3O2)2 for MICP experiments protect building materials from corrosion caused by chloride (Zhang, Guo, & Cheng, 2015). Although,

most studies in the literature utilizes CaCl2 for MICP studies which have provided sufficient

information required to optimize MICP performance. Also, the use of CaCl2 for MICP process

often results in calcite crystal formation which is the most stable form of CaCO3 polymorphs (Achal & Pan, 2014).

Stability of CaCO3 crystals is critical for soil biocementation and its engineering

properties. However, they determined that CaCl2 was the preferred source because it produced

the most stable form of CaCO3 polymorph (calcite), produced high urease and CaCO3 content.

Scanning electron microscopy (SEM) result showed that CaCl2 and CaO had calcite crystals,

but Ca(C2H3O2)2 and Ca(NO₃)₂ showed aragonite and vaterite which are unstable polymorphs

of CaCO3. Zhang et al., (2015) also reported having aragonite-type crystal structure from

mortal samples treated with Ca(C2H3O2)2, while those treated with CaCl2 and Ca(NO₃)₂ showed calcite structures (hexahedral and acicular crystals). However, contrary to Achal and Pan,

(2014), Zhang et al., (2015) reported that specimen treated with Ca(C2H3O2)2 when compared to other treated samples, under same conditions (urease activity) resulted in higher compressive

strength. Jing and Wu, (2014) performed a comparative investigation on the use of C6H10CaO6 and Ca(C₅H₈NO₄)₂ for biocalcification. Their work showed that cementation solution

containing Ca(C₅H₈NO₄)₂ had higher MICP efficiency than that of C6H10CaO6. Their SEM

analysis revealed that Ca(C₅H₈NO₄)₂ had calcite crystal, while C6H10CaO6 had both vaterite and calcite crystal. Besides, Jing et al., (2014) further showed that Ca(C₅H₈NO₄)₂ could serve as a preferred calcium source because it could not only produce predominantly calcite crystal, however it could increase 50% resistance to carbonation. Optionally, to reduce the effect of

CaCl2 on treated soil, researchers can flush excess water upon the soil after completion of the treatment for removal of unwanted chloride present. Also, optimizing the concentration of cementation reagents would help resolve this challenge. A lot of studies on optimizing the concentration of cementation reagents have been performed to identify the ideal concentrations which could favour the predominant formation of calcite crystals (Anbu et al., 2016; Martinez et al., 2013; Mortensen et al., 2011; Zhao et al., 2014b).

Since cementation reagents are critically essential for MICP process, several researchers have investigated the potential usage of other materials which could replace analytical-grade

urea and CaCl2. Cheng et al., (2014) performed a preliminary investigation on how seawater 2+ could be used to obtain the essential Ca sources needed to induce CaCO3. The prepared artificial seawater was compared with conventional cementation solution that consisted of urea -1 -1 (60 g.L ) and CaCl2 (111 g.L ) before MICP treatment on carried out on sand column. Cheng et al., (2014) reported that despite the high salinity concentration present in the artificial seawater, the ureolytic bacterial cells were able to adapt and still survive and produce urease. They suggested that the presence of yeast extract and high concentration of ammonium sulphate used for cultivation inhibited osmotic effect from the seawater.

The XRD analysis showed typical calcite pattern in both samples, but the treated with seawater also showed the peak of the presence of magnesium carbonate trihydrate which is because the seawater contained magnesium ion concentration. Their UCS test result showed that for an equal amount of crystal precipitates obtained, the strength result with seawater was higher than that treated with typical cementation solution and addition of urea to the seawater did not impede formation of carbonate crystals. However, their preliminary study showed that to obtain strength of 300 kPa, the artificial seawater (85 mL) would be require repeated injections (200 times) onto the soil column. Although, in the presence of urea, 50 repeated

injections will be required to increase the reaction rate to induce CaCO3 precipitations. Interesting, Choi et al., (2016) reported that eggshell mixed (ratio of 1:4) with diluted vinegar

(5%, v/v) could be used to obtain soluble calcium source and serve as alternative to CaCl2 for soil biocementation. Biocemented soil sample were 335 to 392 kPa for UCS test, 1.62 to 6.54 -6 -1 x 10 m.s for permeability test and 4.4 to 8.2% for CaCO3 content, respectively.

These obtained data were similar to the control samples (treated with cementation solution) but smaller than results previously reported by Al Qabany and Soga, (2013) (350 to 1300 kPa) and Ivanov et al. (2014) (700 to 1000 kPa) for calcite contents of 6 to 7%. Choi et al., (2016) suggested that the reason their samples showed lower data when compared to other researchers may be due to different ureolytic bacteria and MICP procedures used. Choi et al., (2017) later showed that calcium ions could also be obtained by mixing limestone from aggregate quarries with acetic acid 7% (w/v).derived from lignocellulosic biomass fast pyrolysis as a cost- effective alternative cementation reagent for MICP treatment. Their soil treatment result −6 -1 showed that the CaCO3 content (5.67 to 8.19%) and permeability (8.17 to 1.52 × 10 m.s ) for the biocemented sand specimen was comparable with previous studies available in the literature. Also, analytical-grade urea is typically used for ureolytic activity and MICP process. However, Chen et al., (2018) recently experimented on the feasibility of replacing synthetic

urea with pig urine for CaCO3 precipitation. Their results showed that pig urine could support

sufficient CaCO3 crystals (43% more when compared with control sample) which allowed decrease in permeability and posoity of the treated soil column. Chen et al., (2018) suggested that pig urine could serve as suitable cost-effective raw material for MICP process that may help reduce ammonia production and prevent environmental pollution.

2.5.3. Cultivation medium Microbial growth nutrients are essential for cellular materials (carbon and minerals) and provisions of other essential energy sources or growth conducive factors (Mitchell & Santamarina, 2005). Similar to every biological entity, microorganism requires a wide range of nutritional components in growth medium which comes in the form of major or trace elements (Al-Salloum, Hadi, et al., 2017). The type of nutrients used for bacterial growth can have a remarkable effect on the outcome of the MICP process. Cultivation medium may favour or inhibit essential biomass, enzymatic activities and biominerals precipitation which are important features that make MICP process efficacious. For most MICP studies, commercially procurable analytical-grade cultivation media such as yeast extract, tryptic soy broth, nutrient broth and luria (or lysogeny) broth are commonly used by researchers for enrichment culturing, enumeration and isolation of various microorganism cells.

Depending on the manufacturer (i.e. HiMedia, Thermo Scientific and MilliporeSigma), these culture media contain varying concentrations of its constituents to effectively support microbial growth and absorption of necessary nutrients. For example, nutrient broth media contain 1.5 to 5 g.L-1 of yeast extract, 5 to 15 g.L-1 of peptone, 5 to 6 g.L-1of sodium chloride, 1 to 3 g.L-1 of beef extract or glucose, and initial pH of 7.4 ±0.2 (at 25°C). Tryptic soy broth media which is also known as soya casein digest medium or tryptone soya broth typically contains 2.5 g.L-1 of dextrose, 5 g.L-1 of sodium chloride, 2.5 g.L-1 of dipotassium hydrogen phosphate, 3 Dipotassium hydrogen phosphate of soya peptone, 17 g.L-1 of pancreatic digest of casein or tryptone and initial pH of 7.3 ±0.2 (at 25°C). Luria broth media (5 g.L-1 of yeast extract, 10 g.L-1 of peptone or casein enzymic hydrolysate, 5 g.L-1 of sodium chloride and initial pH of 7.0 ±0.2 (at 25°C) contains similar contents or concentrations as those in nutrient broth. Lower concentrations of cultivation media are also added as constituents of cementation solution. This is often done to promote or simulate bacterial cells growth during biomineralization test or soil biocementation treatment. Bacterial growth in most soils is often limited due to the lack of organic constituents (Mitchell & Santamarina, 2005). It has been suggested that the type of cultivation medium and its constituent can affect the efficacy of MICP process (Al-Salloum, Abbas, et al., 2017; Al Qabany et al., 2012). De Muynck et al., (2008) revealed that nutritional composition has a profound impact on the morphological

formation of CaCO3 crystals formation.

Their work showed that when mortar specimens were treated with cementation solution containing nutrient broth, calcite rhombohedral crystals were absent when subjected to SEM analysis, but lucidly available when treated with cementation solution which did not have nutrient broth. The presence of proteins in the nutrient broth could tremendously influence the crystal growth pattern by adsorption of protein or organic matter (Hammes, et al., 2003; Kawaguchi & Decho, 2002). Shirakawa et al., (2011) investigated the influence of using two cultivation medium to grow three bacterial strains (Lysinibacillus sphaericus INQCS 414, Bacillus subtilis INQCS 328 and Pseudomonas putida INQCS 113) for biocalcification. The two selected medium were modified B4 medium (1 g.L-1 of yeast extract, 1 g.L-1 of glucose and 5 g.L-1 of calcium acetate monohydrate) and medium 295 (3 g.L-1 of nutrient broth, 12 g.L-1 of -1 -1 sodium bicarbonate and 10 g.L of urea and 7.5 g.L of CaCl2).

Although, the initial pH medium was not mentioned in their study, their results showed that for all bacterial strains, the modified B4 culture medium was better than medium 295 for

CaCO3 precipitation. Their data suggested that for the consumption of calcium ions, 96%, 74% and 28% were attributed to Ps. putida, L. sphaericus and B. subtilis, respectively. Thiyagarajan et al., (2016) also studied the influence of nutrients for the survival of bacteria (Bacillus licheniformis) in cement mortar. Cement mortar cubes prepared with sand and cement (ratio of 1:3) were cast before curing them in luria broth and wastewater medium. Although the constituents of these two cultivation medium used in this study did not mention, the authors showed that there was 35% increase in the compressive strength of the mortar when compared to those cured without the nutrient medium. Williams et al., (2016) investigated the suitability of replacing yeast extract with laboratory-grade alternative nutrient sources (lactose mother liquor, corn steep liquor, meat extract, glucose and sodium acetate) for cement-based materials treated MICP process. Their study showed that a combination of urea-meat extract-sodium acetate served as the most suitable replacement for yeast extract in the growth of S. pasteurii. This alternative medium allowed 75% retardation of cement hydration when compared to other nutrient sources and control sample (yeast extract) without compromising MICP process. Williams et al., (2016) strongly suggested that microbial concrete made with the ureolytic bacterial cells grown in this alternative medium would set quicker than when prepared with yeast extract. Hence, by using urea-meat extract-sodium acetate, it can serve as a more practical cultivation medium that can improve bioconcrete production.

Recently, Kiasari et al., (2019) explored the performance of different nutrient medium when used to stimulate indigenous ureolytic bacteria for soil improvement. These six selected medium were glucose medium (250 mM of CaCl2, 333 mM of urea, 0.01 mM of Ni Cl2 and

5% of glucose), yeast extract medium (250 mM of CaCl2, 333 mM of urea, 0.01 mM of Ni Cl2

and 5% of yeast extract), sodium acetate medium (250 mM of CaCl2, 333 mM of urea, 0.01

mM of Ni Cl2 and 100 mM of sodium acetate), sugarcane molasses medium (250 mM of CaCl2, -1 333 mM of urea, 0.01 mM of Ni Cl2 and 3 g.L of molasses), Mol.2 YE.4 medium (250 mM -1 -1 of CaCl2, 333 mM of urea, 0.01 mM of Ni Cl2, 4 g.L of yeast extract and 2 g.L of molasses), -1 Mol.4 YE.2 medium (250 mM of CaCl2, 333 mM of urea, 0.01 mM of Ni Cl2, 2 g.L of yeast -1 extract and 4 g.L of molasses) and reagent medium (250 mM of CaCl2, 333 mM of urea and -1 0.01 mM of Ni Cl2, 4 g.L ). Analysis for treated soil specimens for shear strength ranged

between 91.1 to 140%, UCS ranged between 430 to 450%, and CaCO3 content ranged between 6.8% and 13.8%, respectively. The analysis from Kiasari et al., (2019) indicated that despite the improved biocementation which occurred, samples which were subjected to yeast extract medium had the most effective treatment for stimulation of native ureolytic bacterial cells when compared with other nutrient medium and control.

For large scale bacterial production it is necessary to find an inexpensive substrate that supports a good level of urease activity (Cuzman, Richter, Wittig, & Tiano, 2015). The disadvantages of using the standard microbiological medium for bacterial growth had to do with the operating cost of MICP technology at the commercial scale because the cost associated with supplying the necessary organic matter (i.e. yeast extract) would surely lead cause significant economic limitations (Joshi, Goyal, & Reddy, 2018a). Several researchers revealed that to curb the dependency of ureolytic bacterial cells on standard analytical-grade media, other alternative nutrient sources can be used for bacterial production due to their availability and relatively low-cost. Whiffin, (2004) was one of the earliest MICP researchers to study the possibility of replacing costly cultivation media which could be obtained at low or no cost for large-scale bacterial production. Whiffin, (2004) studied how five protein sources (corn steep liquor, Torula yeast, vegemite, brewery waste yeast and sludge biomass from wastewater treatment processing plant) could replace laboratory-grade yeast extract media (Beckson Dickenson) which was procured at US$204 per kilogram. The alternative protein sources were procured between US$2–US$36 per kilogram) except for sludge biomass which was obtained for free.

Results from the author showed that while brewery waste yeast and the sludge biomass could not support the growth of S. pasteurii, vegemite, on the other hand, showed comparable result (urease activity) with laboratory-grade yeast extract. Furthermore, Whiffin, (2004) suggested that by using a cultivation medium containing vegemite (13.5 g.L-1) and acetate (150 mM), the urease activity rate was increased by 44%. Achal et al., (2009) offered the use of industrial effluent from dairy industry (lactose mother liquor) to replace the standard medium (nutrient broth and yeast extract) for biocementation. The three studied medium all contained -1 5 g.L of NaCl, 2% (w/v) of urea, 25 mM of CaCl2 and initial pH were adjusted to 6.5. However, for lactose mother liquor 10% (v/v) was used, while for nutrient broth (8 g.L-1) and yeast extract (1 g.L-1) were used. Achal et al., (2009) showed that urease activities (412 U.mL- 1 for nutrient broth medium, 366 U.mL-1 for yeast extract medium and 353 U.mL-1 for lactose

mother liquor), the CaCO3 content (28.4% for nutrient broth medium, 26.3% for yeast extract medium and 24.0% for lactose mother liquor) and compressive strengths (27.9 MPa for nutrient broth medium, 27.2 MPa for yeast extract medium and 26.3 MPa for lactose mother liquor) for all samples were comparable. Although it was not mentioned if lactose mother liquor was obtained for free or at a certain cost, their result, however, indicated that this medium could also serve as an alternative protein source for ureolytic bacterial growth and possibly reduce MICP cost.

Similar to the previous investigation, Achal et al., (2010) elucidated that corn steep liquor (1.5%) can serve as a replacement for nutrient broth and yeast extract media. After carrying out their experiments in conditions similar to Achal et al., (2009), their obtained results indicated that urease activities (412 U.mL-1 for all medium), protease activity (38.3 U.mL-1 for nutrient broth medium, 36.9 U.mL-1 for yeast extract medium and 44.2 U.mL-1 for corn steep

liquor), CaCO3 content (38.3% for nutrient broth medium, 36.9% for yeast extract medium and 44.2% for corn steep liquor and compressive strengths (28 MPa for nutrient broth medium, 27.2 MPa for yeast extract medium and 27 MPa for corn steep liquor) for all samples were comparable. Cuzman et al., (2015b) tested a variety of alternative nutrient sources (dairy, poultry and brewery industries) on the growth of S. pasteurii and urease activity in 2 L bioreactor. Their study showed that when the bacterial cells were grown in whey (by-product from dairy industry) growth medium, it had about 80 % more urease activity when compared to other alternative sources and standard medium. Cuzman et al., (2015b) suggested that the production cost of using this medium would be ten times lesser than that of vegemite which was used by Whiffin, (2004).

The researchers were also able to successfully scale-up ureolytic bacterial cells using unsterilized whey nutrient medium containing technical-grade urea (20 g.L-1) in a 5 L bioreactor at 24°C. The initial pH medium was not adjusted and at the end of the incubation period, the measured urease activity for S. pasteurii was 0.5888 mM urea hydrolysed.min-1. Cuzman et al., (2015b) also noticed that when the incubation temperature was increased to 30°C, the urease activity (1.6992 mM urea.min-1) improved at the same urea and proteins concentration. Yoosathaporn et al., (2016) studied the possibility of using chicken manure effluent medium (25%, v/v) as an alternative nutrient source to cultivate ureolytic bacteria for biocalcification of cement cubes. Performance of the chicken manure effluent medium which -1 contained 20 g.L of urea and 25 mM of CaCl2) was compared with three conventional media. These media were nutrient broth (3 g.L-1 of beef extract, 5 g.L-1 of peptone, 20 g.L-1 of urea -1 -1 and 25 mM of CaCl2), tryptic soy broth (30 g.L of tryptic soy broth, 20 g.L of urea and 25 -1 -1 mM of CaCl2), and modified production medium (0.5 g.L of yeast extract, 2.6 g.L of -1 -1 -1 - (NH4)2SO4, 1 g.L of MgSO4.7H2O, 0.032 g.L of NiSO4.7H2O, 2.4 g.L of KH2PO4, 5.6 g.L 1 -1 of K2HPO4, 20 g.L of urea and 25 mM of CaCl2). Analysis from Yoosathaporn et al., (2016) results showed that biomass production, pH, urease activity, water absorption and compressive strength for samples performed with chicken manure effluent showed comparable performance with the standard medium and in some cases had better results.

The alternative medium was able to support bacterial growth (5.91 log cfu.mL-1), pH (9.76), urease activity (16.756 U.mg-1), water absorption (22.52) and compressive strength (29.81 MPa). Furthermore, cost analysis showed that using chicken manure effluent (US$ 0.71 per litre) was more than 80% cheaper than the standard cultivation medium (US$ 6.4 per litre for tryptic soy broth medium, US$ 0.34 per litre for nutrient broth and US$ 2.9 per litre for modified production medium) used in their study. Recently, Joshi et al., (2018a) investigated the possibility of replacing the costly laboratory-grade nutrient broth medium with corn steep liquor (industrial by-product) during MICP treatment and determine if it permits remediation of cracks in bioconcretes repairs. The disadvantages of using the standard microbiological medium for bacterial growth had to do with the operating cost of MICP technology at the commercial scale because the cost associated with supplying the necessary organic matter (i.e. yeast extract) would surely lead cause significant economic limitations. Joshi et al., (2018a) prepared concrete mix with the sand, cement and bacterial cell (4 × 108 cells.mL-1) which were grown in corn steep liquor medium (1.5%, v/v) and nutrient broth (25 g.L-1) for comparison.

Both medium also contained urea (2%, w/v), 25 mM of CaCl2 and initial pH were adjusted to 7.5. The bioconcrete specimens were subjected to different curing methods (i.e. submerged or sprayed in (or with) cultivation medium and bacterial cells for 28 days. Their result showed that the use of this alternative medium for bacterial cultivation and MICP treatment did not affect the properties of bioconcrete produced. Also, their data suggested that bacterial spray treatment with the corn steep liquor seem more suitable especially when considering potential field-scale MICP work.

2.5.4. pH The pH plays a vital role for bacterial transportation and adhesion to promote homogenous distribution of CaCO3 content and compressive strength of the treated soil. It is imperative to investigate how optimum pH conditions could be useful for obtaining better soil solidification

process (Harkes et al., 2010; Imran et al., 2019). The influence of pH on CaCO3 formation is

mainly due to CO2 stability at an elevated pH level (Mamo & Mattiasson, 2019). Both acidic and basic conditions impact the outcome of MICP treatment which may result in lower compressive strength when compared to samples treated in neutral pH condition (Mujah, Shahin, & Cheng, 2016). It has been suggested that MICP favours a pH environment ranging from 6.5–9.3 (neutral to basic conditions) (Fujita et al., 2004). Given the importance of pH in influencing the MICP process, researchers now tend to determine the optimum pH conditions which will favour the performance of the selected bacterial species used in their experiments. Several investigations have indicated that pH 8–9 are the optimum condition for urease activity; these alkaline pH conditions are vital for ammonia production via ureolysis (Omoregie et al., 2017; Stabnikov et al., 2013; Stocks-Fischer et al., 1999). However, other studies have shown that acidic pH and more extreme alkaline pH conditions can also support efficient MICP. Seifan

et al. (2017) investigated the effect of alkaline pH (9–12) on the production of CaCO3 and the bacterial cell concentration under controlled-pH batch conditions in 3 L laboratory-scale bioreactor at 35oC and 150 rpm for 180 h. Their work showed that the bacteria could grow in an alkaline pH environment but the cell concentration decreased as the pH was increased. They also showed that cell viability reduced more than 2.5–fold at pH 10–12 when compared to pH 9. When the initial pH of the medium in the bioreactor was set at 9, bacterial growth increased 3–fold compared to uncontrolled conditions.

Their work also showed induced CaCO3 was 37% greater higher pH level. Kim et al., (2018) studied the optimal conditions of Staphylococcus saprophyticus and S. pasteurii for -1 CaCO3 precipitation. Both microbes were injected into to solution containing urea (1 g.L ) and -1 CaCl2 (14 g.L ) with initial pH of the medium varying between 6 and 10. Their study indicated that when both ureolytic microorganisms were incubated at 30oC, alkaline pH produced the

greater amount of CaCO3 precipitates. Kim et al. (2018) noticed that changes in initial pH had less influence on the productivity of S. saprophyticus, while it had a stronger influence on S.

pasteurii. Their data showed that the precipitation difference in measured CaCO3 crystals at different initial medium pH was 25% and 60% for S. saprophyticus and S. pasteurii, respectively. However, a different study on the influence of different initial pH (8–11) on MICP showed a contrary outcome (Deng & Wang, 2018). Deng and Wang (Deng & Wang, 2018) tested the performance of S. pasteurii (ATCC 11859) during MICP treatment of coral sand. Their results showed that changes in different initial pH did not significantly influence the outcome of MICP. The bacterial cell densities were all above OD of 1.0 after 24 h incubation at 30°C with shaking (180 rpm). This implied that different bacterial species would have different behaviour when cultivated in a media containing various pH levels. While pH may influence MICP, regulating the pH of cultivaiton media may not be a critical factor during field-scale experiment since ureolytic bacteria are capable of adapting to unfavourable pH environments. Indeed, in a recent study performed by the current authors (Omoregie, Palombo, Ong, & Nissom, 2020), large-scale bacterial cultivation was performed in a 3000 L custom- made reactor tank and the initial pH of the medium was not regulated. This did not affect the the growth performance of the bacteria, nor did it hinder the cells from producing sufficient urease and CaCO3 required for soil biocementation.

2.6. Soil biocementation The use of MICP process for soil biocementation has been successfully proven through scientific and industrial investigations. While there are numerous potential and on-going utilization of MICP technology, soil biocementation for geotechnical engineering application has become the most widely reported exploration. Biocementation has been at work in nature since time ancient through biodeposition of sand over a long period (Mujah et al., 2017). Some examples of natural occurrence that may be associated with biocementation are cliffs by the sea, karsts in cave and pinnacles in the desert.

However, the biochemical and microbial activities which facilitate the biocementation to occur natural manner requires a long period. In soil biocementation through the ureolysis- driven process, the rate of biomineralization is hastened, thus allowing biocalcification to be completed under a considerable short period. Through MICP, precipitated cementitious crystals link soil particles together by effective bridging that happens at a concentration contact point as shown in Fig. 2.6 (Mujah et al., 2017). As the cementation media permeates into the soil specimens, bonding in the sand particles improves the engineering properties (Zhao, et al.,

2014a). The enhancement of these engineering properties of soil through CaCO3 crystal precipitation at limited GHG production shows that soil biocementation is surely a green technology to be considered in modern building and development era (Charpe, Latkar, &

Chakrabarti, 2019). The efficiency of CaCO3 precipitation relies fervently on certain key parameters which include biological (enzymatic activity), chemical (calcifying chemical solution) and physical (nature of soil) (Esnault-Filet, Gadret, Loygue, & Borel, 2012). This is because the bacterial strains, cementation reagents, and soil types can influence the outcome of biocementation. For example, biocementation on clay would experience less cohesion than sand grains, thus resulting in limited biocalcification or biominerals precipitated within the soil matrix. Hence, different conditions need to be considered when carrying out soil biocementation. It is not surprising that MICP would not be efficient on clayey soil because its particles have a net negative charge which could influence the local dynamics of the carbonate precipitation. Additionally, pore size in the clayey soils is much lower than sandy soils. This will definitely inhibit penetration, movement and sufficient crystal formation for solidification to occur. However, it is worth noting that numerous soil types (including clay) have been experimented on for soil improvement through MICP process as shown in Table 2.2.

During biocementation process, in most cases, bacterial cells (liquid or powder form) gets encapsulated by the crystal precipitation and restriction of insufficient amount of available nutrient results in the death of the entire bacterial cell (Ariyanti, 2012). It has been suggested that the use of urease-containing cell-free supernatant rather than the whole ureolytic microorganism for soil biocementation lowers the risks of spreading pathogens to the environment or altering the microbial diversity at the treated region (Abdel-Aleem et al., 2019). Most studies utilize whole ureolytic microorganism for MICP studies but only a few have considered using commercially available urease or cell-free urease solution (Abdel-Aleem et al., 2019; Carmona, Oliveira, & Lemos, 2016; Yasuhara, Neupane, Hayashi, & Okamura, 2012).

However, it is essential to clarify that this depends on the type of ureolytic bacteria used, for example, some microbes (Pseudomonas azotoformans and Pseudomonas aeruginosa) used for MICP may be pathogenic, thus non-pathogenic bacteria such as S. pasteurii is often preferred. It has also been suggested that the cell-free enzyme is suitable for soil biocementation because it avoids the complex storage process which requires special environmental conditions (Carmona et al., 2016).

Biocementation studies from cell-free urease have also shown that UCS and permeability tests conducted resulted in improved mechanical conditions of the soil. For example, an investigation by Yasuhara et al., (2012) showed that their soil specimen using cell-free urease solution resulted in UCS ranging from 400 kPa to 1.6 MPa, and the permeability of the improved soil samples reduced by more than one order of magnitude. Hence, the use of a cell- free enzyme can serve as an alternative to the whole microorganism for soil biocementation investigations. This may also be very limit transportation liquid cells to the site, prohibit the need for cultivation and ease mixture with soil. Thus, it would be interesting to see more research studies on the use of powdered bacterial cells than broth culture cells for MICP. Although, companies such as Soletenache Bachy, have shown on commercial-scale that both the use of liquefied or powdered bacteria can be used for soil biocementation, unfortunately. One of the main reasons most MICP researchers utilize whole-cell ureolytic microorganisms for soil treatment is due to its cost-effectiveness. Commercially procurable urease typically extracted from jack bean (Canavalia ensiformis) which could be in powder or liquid form is extremely expensive and may range between $43.6 to $1260. Since the bacterial cells mostly

act as a driver for production of urease and precipitation of CaCO3 crystals, it is therefore adequate to use whole ureolytic microorganism for numerous MICP applications.

Biocementation has been shown to have the capability of replacing chemical admixtures and other environment harming substances for soil solidification, thus making it a sustainable cementitious treatment method (Charpe et al., 2019). The controlled approach is necessary to attain successful biocementation outcome at large-scale because it requires the addressing of microbial, chemical and geotechnical parameters (Esnault-Filet et al., 2012). Hence, to consistently assess biocementation improvement in variable field conditions, more investigations must be carried out to identify and characterize the limitations and optimum conditions which impede or favour biocementation (Gomez, DeJong, & Anderson, 2018).

While the effectiveness of biocementation has been extensively demonstrated at the laboratory conditions (i.e. centimetre-scale), only a few large-scale and field-scale trials have been demonstrated to understand the biogeochemical process of MICP at meter scale (Gomez, et al., 2020). Some of the investigations (large-scale and field-scale) by different research groups have been summarized in Table 2.3. Biocementation treatment process can be achieved at field-scale on different types of soil, irrespective of the particle sizes, environmental conditions or concentration of cementation reagents used (Mortensen et al., 2011). The demonstration of soil biocementation at meter scales are relevant for practical soil improvement because they provide critical knowledge on the biogeochemical kinetics which are associated with the reaction, bacterial behaviour and identity practical limitations to the implemented MICP techniques (Gomez et al., 2020). Hence, the need to promote conditions which will inspire future in-situ field-scale MICP investigations for soil biocementation is highly needed. Also, since field-scale MICP trials are costly and can be time-consuming, some researchers have utilized models to bridge the gap of laboratory-scale MICP process to practical implementation. These researchers have used computational simulators (i.e. reactive transport model) to study the behaviour of bacterial transport and attachment, porous medium and solution, enzyme activity and CaCO3 precipitation to promote successful soil biocementation (Cunningham et al., 2019; Dupraz, Parmentier, Ménez, & Guyot, 2009; Minto, Lunn, & El Mountassir, 2019)

Fig 2.6: Schematic diagram showing the effective bridge formation (Mujah et al., 2017). Reprinted with permission from Taylor & Francis.

Table. 2.2: Type of soils obtained from various locations treat with MICP process

Soils Sources Soil properties Studied aspects Remarks Authors

Peat soil Sakarya, pH (4.5 to 6.5); organic carbon Bacteria counting, soil pH values reached 9.3 for organic soil Sidik et al., Turkey (20-30%); Water keeping biocementation, pH samples after 12 h of MICP treatment (2014) capacity (85 to 95%); Natural analysis, permeability and a 10-fold reduction in water content (256%); Liquid test, calcium carbonate permeability was obtained (5.2 X 10-3 limit (125%); Specific gravity content and cm.s-1 to 4.5 X 10-4 cm.s-1). The -3 (1.97 g.cm ); silt (15%); clay microstructure analysis. contents of CaCO3 precipitates in the (25%); sand (25%); organic treated peat soil was lower than that in

material (60%); ash content treated sand. Also, CaCO3 precipitates (0.4%). in the peat specimen were not uniform over the specimen length.

Silty soil Poznan´, Silty fraction content (50 to Soil biocementation, The biodeposited specimen resulted in Grabiec et Poland. 69%); permeability test, traxial increased shear strength (152 to 275 al., (2017) specific gravity (2.66 g.cm-3); compression test, test, kPa) in the triaxial compression test natural moisture content (17%); bacterial survival test due to biocementation of dense silt. In liquid limit (32.5%); plastic limit and moisture test. addition, changes occurred to the (22.91%); plasticity index optimum moisture (1.775 to 1.765 (9.59%). g.cm-3) and maximum volumetric

density (14.5% to 13.5%) after MICP treatment.

Sandy soil Giza, Egypt Specific gravity (2.88 g.cm-3); Soil biocementation, The use of injection with a fixation Abo-El- pH (8.18); total dissolved solids water stability test, solution during MICP treatment was Enein et al., (449.5 ppm); Cl (39.5 ppm); unconfined compressive more effective than the injection (2013)

SiO2 (96.12%); Fe2O3 (0.44%); strength and without fixation solution. The increase

Al2O3 (1.05%); CaO (1.33%) direct shear test, point in calcite deposition led to increased

and CaCO3 (2.39%). load test, bulk density, resistance to sand deterioration and the slake use of non-sterilized medium had no durability index test, negative effect on S. pasteurii biomass cementing material and biocalcification. ratio content, XRD analysis and microstructure analysis.

Clayey soil Coastal area Specific gravity (2.70 g.cm-3); Biocementation and The bioencapsulation had an increase Ivanov et of water content (55%); pH (7 ±8); bioencapsulation, pH in the strength (2 MPa) for the treated al., (2015) Singapore. Al (7 ±2%); Si (18 ±4%); O (65 analysis, measurement specimen. However, it was noticed ±7%); C (5 ±1%); Cl (2 ±1%); of calcium that these strengths decrease with the Ca (1%); Mg (1%); Fe (1%) and concentrations, strength increase in the size of the aggregate K (1%). test, microstructure when the size is greater than 5 mm.

analysis and atomic force microscopy.

Calcareous Xisha Island, specific gravity Measurement of urease Increase in cementation solution or Liu et al., soil South China (2.75 g.cm-3); mean grain size activity and calcium sample volume resulted in improved (2019) Sea (D50, 0.36 mm); coefficient of concentration; soil unconfined compressive strength, the uniformity (2.36); minimum biocementation; splitting tensile strength of the treated void ratio (1.27); maximum unconfined specimens and observed the presence

void ratio (1.75); compression and of CaCO3 crystals determined via calcium (93.70%); splitting tensile test; microstructure analysis. magnesium 2.94%); strontium triaxial compression (1.92%); sodium (0.44%); tests and microstructure phosphorus (0.34%); sulphur analysis. (0.29%); and aluminium (0.17%).

Coarse soil Center of Clay (28.3%); silt (50.0%); sand Soil biocementation; Increased strength due to Venda Portugal (21.7%); Specific gravity (2.75); unconfined compressive biocementation with increases in the Oliveira and

Liquid limit (52.5%); Plastic strength tests; calcium amount of CaCO3 precipitate, with Neves, limit (22.3%); Plasticity index carbonate content; greater significance when the content (2019)

(30.2%) and pH (5.06). microstructure analysis; of CaCO3 is lower than 0.5%. XRD analysis.

Lateritic Espirito do A stiff yellowish brown silty Soil permeability The coefficient of permeability for the Smith et al., soil Santo, Brazil. clay with frequent quartz testing and carbonate lateritic soil specimen was (2017) (according to BS5930:2015); content. successfully reduced (1.15 × X 10-7 classified as a low plasticity clay m.s-1 to 1.92 X 10-8 m.s-1) which was (according to BS1377-2:1990); an 83% reduction in permeability. liquid limit (30.5%); plastic limit However, it could not be determined if (19.9%); plasticity index the changes in soil mass were due to

(10.6%); maximum dry density CaCO3 crystals or simply S. pasteurii (1.7 Mg.m-3); optimum moisture biomass aggregation. content (16%) and particle density 2.65 Mg.m-3).

Table 2.3: Previous large-scale and field-scale investigations on biocementation for soil improvement

Specimen column Microbial inoculant Treatment condition Summary of findings Authors

Sandy soil (relative Bacillus sphaericus 18 L of bacterial suspension (3L Ammonium measurement of Cheng and density of 1.60 (MCP-11) which was for initial infiltration) and 96 L of effluent showed 90% urea Cord- -3 g.cm and porosity cultivated in medium (20 cementation solution (1 M CaCO3 hydrolysis, while Schmidt Ruwisch, of 40.8 %.) was g.L-1 of yeast extract, 0.17 and urea) were percolated into the Hammer test showed (2014) placed in a M of ammonia sulphate, soil column and incubated at room considerable strength (1 - 4 MPa) cylindrical 0.1 mM of nickel chloride temperature. and reasonably homogeneous container (450 mm and initial pH of 9.25) had CaCO3 content (300 mm in in diameter and urease activity of 10 mM diameter and 200 mm in depth). 500 mm in height). urea.min-1.

The loose sandy S. pasteurii (ATCC 1376). Bacterial cultures in 4 L Sandstone-like crust (0.32 to 2.5 Gomez et soil on site which which was cultivated in containers (treatment on only day cm) was observed, a significant al., (2015) had four test plots medium (20 g.L-1 of yeast 1), nutrient broth (3 days increase in dynamic cone (2.4 m x 4.9 m) extract, 10 g.L-1 of treatment) and 379 L of penetrometer resistance were established at ammonium sulphate, 3.4 cementation solution (15 to 60 measurement (depths more than 5 -1 -1 the project g.L of trisma acid and g.L of urea and 13.9 to 55.5 cm) and CaCO3 content (2.1%) -1 -1 location. 13.1 g.L of trisma base) g.L CaCl2) were injected (flow were reported. had a cell density of 108 rate of 19 L.min-1) into the loose cells.L-1. soil for 20 days.

3 + 0.15 m soil S. pasteurii (ATCC strain All soil columns (1-5) were Increase in NH4 concentrations Lee et al., specimens 11859) used in this study injected (flow rate of 400 mL.min- (1 to 5 mM were obtained, with (2019a) 1 + (porosity of 0.30 – (for only column 3) had a ) with 76 L of augmentation final cumulative NH4 removal

0.40% and relative cell density of 9.36 × 107 solution (50 mM of urea, 100 mM (97.9% and 99.8%) and the shear

-1 density of 55 – cells.mL . of NH4Cl, 42.5 mM of C2H3NaO2, strength results which were

67%) were places 0.04 to 0.2 g.L-1 of yeast extract, measured along the lengths of the five 3.7 m long 1.28 g.L-1 of sodium hydroxide and columns of the treated soil. hollow steel initial pH of 8.4 to 9.0) to stimulate columns with native ureolytic bacteria and square cross- cementation solution (250 mM of sections (0.2 m by CaCl2, 250 mM of urea, 12.5 mM

0.2 m). of NH4Cl, 42.5 mM of C2H3NaO2,

0.02 to 0.2 g.L-1 of yeast extract,

and initial pH of 8.4 to 9.0) to

initiate biocalcification except for

column 3 (contained S. pasteurii, 9

g.L-1 of yeast extract, and initial pH

of 7.0).

A tank (height of S. pasteurii (ATCC #1376) The tank was subjected to injection The post-treatment shear wave Gomez et 0.5 m and diameter which was cultivated in of 107 L augmentation solution velocity measurement showed al., (2020) of 1.7 m) contained medium (20 g.L-1 of yeast (0.1 g.L of Trisma acid, 13.1 g.L-1 high stiffness near primary Yolo loam extract, 10 g.L-1 of of yeast extract, 12.5 mM of injection location (970 m.s-1), the -1 (plasticity index of C2H3NaO2, 3.4 g.L of NH4Cl, 42.5 mM of C2H3NaO2 and tip resistance measurement at 18%), concrete Trisma acid and 13.1 g.L- 3.5 x 107) on day 1, 107 L pre- mid-depth was 25.8 MPa and 1 sand (relative of Trisma base) had a cementation solution (same CaCO3 content achieved was up density of 45%), concentration of 3.5 x 107 contents used for augmentation to 5.1% by mass. and Monterey 12- cells.mL-1. except no bacterial cell) on day 2, 20 sand (relative 161 L of cementation solution density of 43 - (same contents used for pre- 51%). cementation but includes 250 mM

of CaCl2) for 7 days treatments and 476 L of artificial groundwater

solution (1.75 mM of CaCl2, 0.04

mM of KNO3, 0.045 mM of

MgSO4, 0.04 mM of NaNO3, 1.10

mM of NaHCO3 and 0.06 mM of

KHCO3) on day 11.

A cylindrical tank S. pasteurii used in this Using a flow rate of 0.85 PV.h-1, The results suggested that similar Gomez et (diameter of 1.7 m study had a concentration 208 L of stimulation solution ureolysis rates were obtained al., (2019) and height of 0.5 of 3.5 × 107 cells.mL-1. (350 mM of urea, 12.5 mM of from the biocemented treated m) contained 0.3 m NH4Cl, 42.5 mM of C2H3NaO2, tanks and calcite distributions thick layer of dry- 0.1 g.L-1 of yeast extract and initial ranged between 85 to 874 mol.m-3 pluviated poorly- medium pH of 7.3) or for the stimulation tank (native graded soil (3% for augmentation (S. pasteurii) was microorganism) and 169 to fine content and 43 done once, 312 L of injection of 851 mol.m-3 for the augmentation - 51% for relative cementation solutions (identical to tank (S. pasteurii). density). the stimulation solution but included 250 mM of CaCl2) was performed for 5 days, while 936 L of artificial groundwater (40 μM of KNO3, 450 μM of MgSO4, 1.75 mM of CaCl2, 40 μM of NaNO3, 1.1 mM of NaHCO3, and 60 μM of KHCO3) were injected into the tanks for 3 days before curing.

A concrete Sporosarcina pasteurii, 5 m3 of the bacterial suspension The results showed that seismic van Paassen container (8.0 m x (DSM 33) cultivated in was injected (flow rate of 0.3 measurements had significant et al., 5.6 m x 2.5 m) was growth medium (20 g.L−1 m.m−1), followed by 5 m3 of 0.05 increase in shear- wave velocity (2010) −1 -1 −1 filled with 2.25 m of nutrient broth, 10 g.L mol.L of CaCl2 to enhance (118 to 395 m.s ) during of poorly-graded of NH4Cl, 10 µM of NiCl2 bacterial fixation, after which 96 treatment, distribution of CaCO3 sand (dry density and initial pH of 8.5 had a m3 of cementation solution (1 M of content varied from 0.8 to 24% of −3 of 1,560 kg m ). maximum urease activity urea and CaCl2) for 16 days and the total dry weight and the UCS of 1.1 mol.urea L−1.h−1. flushed with 30 m3 of tap water. tests ranged between 0.7 to 12.4 MPa.

2.7. Various techniques used for soil biocementation

2.7.1. Surface percolation method Soil biocementation treatment by surface percolation method allows the introduction of cementation fluid and bacterial cultures into soil matrix through simple pouring or spraying approaches. Soil biocementation treatment through spraying the cementation solution and bacterial cells onto the soil surfaces is not commonly used, however, recent researchers are adopting this approach for soil for production of carbonates crystals and soil biocementation (Ivanov, Stabnikov, Stabnikova, & Ahmed, 2019; Maryam, 2014; Mohebbi, Habibagahi, Niazi, & Ghahramani, 2019; Stabnikov, Ivanov, & Chu, 2016). Gravitational flow and capillarity help transport the solutions via surface percolation onto the surface of the soil columns with a depth of 2 m with ease (Cheng & Shahin, 2016). The main advantage of using this method for soil solidification is that the injection of cementation solution and bacterial culture does not require heavy machinery due to free-draining of fluid movement (Mujah et al., 2017). Hence, this makes surface percolation a cost-effective, simple and practical approach treatment method (Omoregie et al., 2017). Cheng and Cord-Ruwisch, (2012), were one of the earliest researchers to report on the efficacious potential of using surface percolation method for soil biocementation. Their investigation compared the use of surface percolation method as an alternative to submerged flow treatment method previously used by Whiffin, (2004) (Fig 2.7).

Fig. 2.7: Schematic diagram showing different soil biocementation process (Cheng & Cord- Ruwisch, 2012). Reprinted with permission from Elsevier.

Cheng and Cord-Ruwisch, (2012), showed that by alternating either ureolytic bacteria (L. sphaericus) or cementation solutions (1 M of CaCl2 and 1 M of urea), it allowed bacterial cells immobilization over the full length of 1 m pure silica sand column. The UCS and CaCO3 content results for column subjected to surface percolation treatment were 390 KPa and 0.12 g.cm-3, respectively, while that of submerged flow treatment were 340 KPa (UCS) and 0.14 -3 g.cm (CaCO3 content). Their next study showed that this treatment method could also be scale-up large soil column (Fig. 2.8) (Cheng & Cord-Ruwisch, 2014). The result indicated that the treated soil (2 m depth) had UCS of 850 to 2067 kPa. Also, results obtained from the CaCO3 content and mechanical properties confirmed that the surface percolation method is particularly suitable for coarse sand and gravel materials with high permeability. To further explore the practical suitability of this MICP treatment method, Cheng et al., (2017) investigated the possibility of using in-situ enrichment culture of soil ureolytic bacteria to stimulate in-situ urease activity enrichment and soil biocementation. Their results (Fig. 2.9 and Fig. 2.10) showed that ureolytic microorganisms could be activated to produce sufficient urease activity to allow non-bioclogging formation in the treated soil columns (1000 mm). At the end of the investigation, experiments from Cheng et al., (2017) showed UCS of 850 to 1560 kPa for coarse sand and 150 to 700 kPa for fine sand.

Fig. 2.8: Results of a sandy soil sample subjected to MICP treatment via surface percolation (Cheng & Cord-Ruwisch, 2014). Reprinted with permission from Taylor & Francis.

Fig. 2.9: Chart showing the effect of different medium on urease activity of enriched soil ureolytic bacteria (Cheng, Shahin, & Cord-Ruwisch, 2017). Reprinted with permission from Taylor & Francis.

Fig. 2.10: Chart showing the UCS and CaCO3 content of treated soil done with in-situ grown ureolytic bacteria (Cheng, Shahin, & Cord-Ruwisch, 2017). Reprinted with permission from Taylor & Francis.

2.7.2. Injection method Soil biocementation through injection method is the most widely preferred MICP treatment, although it often requires mechanical assistance such as hydraulic system to transport the cementation solutions and bacterial cells within the soil matrix. This technique is most suitable for large-scale biocement experiments (Fig.2.11 and Fig. 2.12) which requires augmentation or injection of bacterial cells and cementation solutions (Gomez et al., 2016). Besides, it is also suitable for stimulation treatment approach which tends to often omit to introduce exogenous bacterial cells into the soil, instead, it stimulates indigenous microorganisms to precipitated biominerals by feeding it with growth nutrients and cementation solutions via injection. While other biocementation treatment methods are commonly carried out in small-scale columns (e.g. polyvinyl chloride), injection treatment method has however been successfully applied unto numerous soil specimens prepared in large tanks. This is because injection treatment methods can easily improve soil layers at a deeper depth. This treatment method is often selected because it has the utmost chance of cemented loose soil particle at further depth. Also, the injection treatment method can be controlled at ease irrespective of the point of injection (i.e. vertical direction or horizontal direction) (Mujah et al., 2017). One of the issues affecting this treatment method is the formation of bioclogging at injection points which often results in the limited distribution of CaCO3 content and heterogeneous compressive strength. This is because, during MICP treatment, bacterial cells and chemicals react instantaneously at contact points and result in localized cementation at injection regions especially during repeated injection cycles (Cheng & Cord-Ruwisch, 2014). Previous MICP studies performed by Whiffin, (2004) and van Paassen, (2009) on soil solidification via repeated cycles of injection treatment methods on soil columns of 5 m long through vertical flow and 100 m3 sandbox through horizontal flow, respectively. Despite

obtaining promising results, their studies showed heterogeneous CaCO3 content in the biocemented soil columns. This cell concentration was diluted in the applied augmentation solution before introduced into the tank and the chemicals were prepared in a mixing tank before injecting in the large-scale tanks (Fig 2.11) (Gomez et al., 2016). For small-scale injection treatment method, the bacterial cells and cementation solutions are usually prepared in glassware before mechanically injected into the soil columns. To obtain the homogenous and spatial distribution of CaCO3 content during MICP process, proper injection procedures, chemical reactions and pH buffer need to be optimized (Al Qabany et al., 2012; Van Tittelboom, De Belie, De Muynck, & Verstraete, 2010; Warren et al., 2010).

Fig. 2.11: Schematic diagram showing tank specimen subjected to injection treatment method (Gomez et al., 2016). Reprinted with permission from the American Society of Civil Engineers.

Fig. 2.12: Biocemented sandy soil observed after MICP treatment using the injection technique (van Paassen et al., 2010). Reprinted with permission from the American Society of Civil Engineers.

Harkes et al., (2010) reported the use of a two-phase injection procedure (i.e. injection of bacterial culture followed by injection of the cementation solution) previously developed by Whiffin, (2004) for bacterial cell retention and prevention of crystal accumulation around injection points. This injection strategy has the advantage of evading the rapid flocculation and bioclogging of the pore voids near the injection end (Cheng, Shahin, & Chu, 2019). This methodology requires the saline diluted suspension of bacterial cells (OD of 2.88 and urease activity of 3.1 mS.min-1) into soil column which was positioned vertically and immediately followed by a fixation fluid for absorption within the soil grain. The flow rate of 200 mL.h-1

was used to inject 1M equivalent molar CaCl2 and urea. Harkes et al., (2010) were unable to provide information on compressive strength and actual distribution of cementation flow at the end of their study. However, they studied the effluent samples for bacterial activity along the column axis and showed that ammonium production occurred throughout the columns, thus suggesting that even distribution of cementation may have been reached in the entire column. Harkes et al., (2010) further discussed the demerit of utilizing diluted bacterial cells in saline solution. They indicated that the dilution provides the risk of osmotic shock to the bacterial cells and also the use of diluted bacterial cultures would increase the total volume of bacteria injection required to acquire the needed level of urease activity. This may lead to unwanted fluid fixation and bacterial cells being washed out during treatment. To also prevent bioclogging formation at injection points, Kakelar et al., (2016) utilized a sequencing batch mode of injection method (multistep injection) previously suggested by Whiffin, (2004) and van Paassen, (2009). By using a flow rate of 200 mL.h-1, bacterial cells (OD of 2.5 and urease -1 activity of 60 mM urea. h ) and CaCl2 solution (0.05 M) was injected into the soil column. After 24 h, to promote flocculation and bacterial retention in the soil, repeated injection cycles

(3) of bacterial cells and cementation solution (1 M of CaCl2 and urea) were performed. At the end of the experiment, improved calcite precipitation and compressive strength were obtained. Kakelar et al., (2016) suggested that contrary to the conventional injection used, multistep injection was able to minimize the required volume for soil treatment by half and can help to avoid washout of bacterial cells during MICP treatment.

2.7.3. Premixing method Soil biocementation by premixing method requires a mechanical mixture of soil specimen with bacterial cells and chemicals solutions. This method is often used alongside other

treatment methods to enhance even distribution of CaCO3 content. Unfortunately, premixing method is the least favourable MICP method because it may disturb the local soil and may lead to the development of pseudo strength during premixing or cause complication to soil stress history and lead to undesired uncertainties during mechanical testing (Mujah et al., 2017). Whiffin, (2004) and Al-Thawadi, (2008) were some of the earliest researchers to investigate the influence of using the premixing method before injecting reagents to enhance the urease

activity and CaCO3 precipitation rate for strength increment. Al-Thawadi, (2008) suggested that premixture of bacterial cells with soil could result in three-to-four fold increase in the mechanical strength of the treated soil. This is because cells attached to the soil after premix could enhance 40 - 64% retention. The author also stressed that premixing calcium ions with bacterial cells and soil before the injection procedure is commenced could increase the cell retention rate and mechanical strength of the biocemented soil. Cheng and Shahin, (2016)

proposed a novel approach to promote even distribution of CaCO3 content after soil biocementation, by using bioslurry crystal formation (Fig. 2.13) which is prepared by adding

a specific amount of urea and CaCl2 into glassware containing bacterial cell, followed by stirring (600 rpm) for 12 h to obtain the required precipitate.

Fig. 2.13: Photos of bioslurry premixed with soil column before MICP treatment (Cheng & Shahin, 2016) Reprinted with permission from Canadian Science Publishing.

Later, this biolsurry is premixed with the sandy soil followed by injection of cementation solution. Cheng and Shahin, (2016) noted that by using the bioslurry alone for soil treatment without adding cementation solution did not allow biocalcification to occur because it could not provide the necessary bonding force needed to solidify soil grains. However, the addition of cementation solution to soil columns that was premixed with bioslurry crystals resulted in even distribution of CaCO3 content and significant strength (1 MPa). Omoregie et al., (2018) recently studied the performance of premixing method on soil columns that were prepared in paper rolls (height of 95 mm and the inner diameter of 45 mm). Their investigation showed that there was a significant difference on the mechanical properties of soils that had (a) no premix; premixed with only bacterial cells and; premixed with only cementation reagent (1 M), before subsequently subjected to injection treatment and curing for 72 h. They reported that from the visible observation that biocemented soil subjected to no premixing and premixing with only bacterial cells had better uniformed cylindrical shaped (Fig. 2.14). However, all treatment conditions resulted in improved soil strength (212.5 to 430.9 KPa), but the columns which were premixed with bacterial cells had the best strength result (Fig. 2.15).

Fig. 2.14: Biocemented sand specimens subjected to MICIP premixing treatment under different conditions; (A) sand without premix, (B) sand premixed with bacterial culture and (C) sand premixed with cementation reagents (Omoregie et al., 2018).

Fig. 2.15: Surface strength of the treated sand samples from different treatment groups after surface percolation treatment. Group 1 refers to sand columns which did not contain bacterial cells or cementation solution premix prior to treatment; Group 2 refers to sand columns which were premixed with only bacterial culture prior to treatment; and Group 3 refers to sand columns which were premixed with only cementation reagents (urea and CaCl2) prior to treatment (Omoregie et al., 2018).

Hence, premixing bacterial cells with soils before injection treatment method may also be a preferred means of improving the calcite content or compressive strength of the soil. The premixing is good to making biocement soil columns to improve cell attachment or distribution within the soil however, this method does not be suitable for practical implementations. Premixing can also be suitable for crack repairs or cementation or production of bio-paver blocks. However, the injection method is the realistic method compared to the premixing method. Injection method can be applied to any soil layers without removing existing soil bodies. However, the bioclogging formation and uneven distribution of CaCO3 contents within the soil matrix are issues to take into consideration. Thus, it is important to ensure bacterial cells are well attached to soil particles before MICP treatment, to avoid unwanted removal of the bacterial cells during injection or surface percolation treatments.

When cells are poorly attached to the soils, it will be easily washed out especially when cementation solution is injected continuously or with high pressure (Al-Thawadi, 2008).

Omoregie et al., (2018) reportedly premixed CaCl2 powder with the sand column but did not try dissolving it in water before usage. Hence, this may have influenced the formation of bulk precipitates within the soil matrix after treatment and caused disintegrated biocemented

columns. It would have been interesting to see the outcome when the CaCl2 solution was premixed with soil before MICP treatment. By premixing bacterial soil in the presence of

bacterial and CaCl2 solution (i.e. 100 mM) would improve bacterial retention (Cheng, Shahin,

& Mujah, 2017). The addition of CaCl2 solution can act as a flocculent and initiate coagulation of bacterial cells before subsequent MICP treatments (injection or surface percolation methods) (Al-Thawadi, 2008).

2.7.4. Submerged treatment method Submerged or immersed treatment method is another alternative approach which has been used to promote the MICP process for soil cementation. This method helps to avoid or resolve the bioclogging formation that occurs at injection points when injection or surface percolation treatment methods are used. It has been expressed that direction of cementation solution and bacterial culture within the soil specimens causes the bioclogging to occur at injection points, thus affecting the engineering properties of the treated soil (van Paassen, 2009; Whiffin, 2004; Zhao et al., 2014a). The submerged method is mostly suitable for in-situ practical problems such as soil stabilisation of submersed or submarine sediments (Cheng & Cord-Ruwisch, 2012). To study the potential of this treatment method, Zhao et al., (2014a) fully submerged soil specimens into a mechanically operated tank reactor containing cementation solution as shown in Fig. 2.16. Before the treatment, the soil (330 g) was first prepared with full contact flexible molds specimens which contained 85 mL of S. pasteurii (OD 0.3 to 1.5). The -1 -1 cementation solution used contained NH4Cl (10 g.L ), nutrient broth (3 g.L ), NaHCO3 (2.12 -1 -1 -1 g.L ), urea (15 to 90 g.L ), CaCl2.2H2O (36.8 to 220.5 g.L ) and initial pH of 6.0. According to Zhao et al., (2014a), the utilized batch reactor did not have any hydraulic gradient to drive the flow through the soil specimen. Hence, when it was expected that by using the submerged method, the ureolysis reaction induced by the bacterial cells resulted in chemicals being lower in the soil specimens. The led to the chemical materials to diffuse from higher concentration area to lower concentration area in the soil specimens.

Fig. 2.16: Schematic drawing of the batch reactor (A) and image of samples after MICP submerged treatment method (B) (Zhao et al., 2014). Reprinted with permission from the American Society of Civil Engineers.

Their reported UCS result ranged between 1.76 to 2.04 MPa and They suggested that submerged method is a more effective MICP process than other known methods. Wen et al., (2019) also utilized the full contact flexible mold previously developed by Zhao et al., (2014a) to serve as a column for their soil specimen (200 g.m-2). The full-contact flexible mold had an opening size of 0.15 mm, water flow rate of 34 mm.s-1 and thickness of 1.5 mm. Their Laboratory-based investigation was carried out to determine the efficiency of using different concentration of cementation solution and multiple treatments via immersing method and multiple treatments could help enhance the mechanical properties of treated soil specimen. Wen et al., (2019) recently reported that under the certain concentration of cementation solution (0.25 to 0.75 M) with repeated treatments, soil biocementation was improved. The strength test shows that their biocemented specimen had UCS of 6400 kPa. Zhao et al., (2014b), later showed that using the submerged method to treat the full-contact flexible mold soil specimens

promoted better MICP improvement to soil column, thus resulting to homogenous CaCO3 precipitation within the soil particles.

2.8. Testing methods often used to evaluate MICP soil treatment It is extremely necessary to access and monitor the performance of ureolysis process for

CaCO3 precipitation. Since MICP evaluation undergoes different physico-chemical and mechanical conditions, several methods have been adopted over the years to verifying the calcification process under in-situ or in-vivo conditions (Al-Salloum, Hadi, et al., 2017). Different genera of ureolytic microorganisms have been employed for numerous MICP applications that provide a similar or different outcome. This review discusses only the major methods that are commonly used to elucidate the efficiency of MICP process specifically from the biotechnological aspect for various potential applications.

2.8.1. Urease activity Urease activity measurement is the most frequently used assay to give a direct indication of bacterial performance during ureolysis process (Al-Salloum, Hadi, et al., 2017). Quantitative determination of bacterial urease activity is done using several analytical approaches. Urease activity through conductivity measurement is carried out after bacterial propagation (Harkes et al., 2010). Conductivity meter is used to determine the electrical changes after bacterial cultures are inoculated into a urea solution (1.5 M) and monitored for 25 ±2 °C (Whiffin, 2004).

The high correlation coefficients usually indicate a positive linkage between the increase in conductivity and urea hydrolysis (Al-Thawadi, 2008; Whiffin, 2004). Determination of urease activity by conductivity measurement is the most common method used in the literature, indicating it is a suitable indication for urease production (Omoregie et al., 2017; Sun, Miao, Tong, & Wang, 2019; Wang et al., 2020; Zhao et al., 2014). However, there are other notable methods which have been successfully used to determine urease production which includes phenol-hypochlorite assay, Nessler assay and colourimetric assay (Berthelot’s reaction) (Chen and Achal, 2019; Cheng et al., 2014; Dhami et al., 2017; Stocks-Fischer et al., 1999). These methods are suitable to study ammonia concentration notwithstanding the presence of calcium

ions or CaCO3 precipitation. During the conductivity test, hydrolyses of urea lead to ammonium and carbonate ions production, thus leading to an increase in the conductivity reading (Sun et al., 2019). The conductivity variation rate is often acquired from the slope of the plotted graph with the inclusion of the dilution factor (Armstrong Ighodalo Omoregie et al., 2017).

2.8.2. Biomass measurement Quantitative determination of microbial biomass as a function of time is very vital both in applied microbiology and biotechnology (Siro, 1985). Bacterial cells behave different during cultivation, especially which can be attributed to their microenvironments. Various factors (i.e. temperature, salinity, initial pH medium, media concentration and constituents) have unequivocally influenced on the growth pattern of bacterial cells which can affect urease activity or biominerals production. Monitoring cell growth would also provide essential information concerning their nutritional and proliferation condition since different genera bacterial species would behave differently (Koch, 2007). More so, if alternative substrates are utilized or optimized as a replacement to some conventional growth media for optimum biomass production, the performance of the bacterial have to be carefully evaluated. There are various classic and modern techniques (i.e. optical microscopy, colony counts, cell number estimation, turbidity measurement, dry weight determination) which are commonly used to determine the cells of microorganisms during or after cultivation (Koch, 2007) (Holm-Hansen & Karl, 1978; Pringle & Mor, 1975). OD or turbidimetry test is often used as a biomass concentration indicator to monitor the performance of MICP bacterial cells based on turbidity measurement (Omoregie et al., 2017).

The OD measurement on growing cell cultures is a standard and simple microbiological method used to quantify important cultivation parameters (i.e. changes in bacterial morphology and biomass production) (Kim & Lee, 2019). The number of bacterial cells is typically determined at a wavelength of 600 nm using an ultraviolet and visible spectrophotometer after the zero point correction (Harkes et al., 2010; Tian et al., 2018). The amount of absorbed light in a bacterial cell suspension can be immediately and directly related to bacteria mass or number (Kim & Lee, 2019). Turbidimetry is the most-widely utilized analytical tool for measurement of bacterial growth in liquid cultures due to its easy, fast and non-destructive feature (Maia et al., 2016). Many MICP studies on ureolytic bacterial cultivation in various growth medium (i.e. with or without urea) have successfully monitored the cell density or concentration using turbidity measurement (Kim & Lee, 2019; Phang et al., 2018; Wu et al., 2019; Xiaohao, Linchang, Tianzhi, & Chengcheng, 2018). Moreover, colony count method is also often used for to monitor the growth behaviour of MICP microorganisms (Arias, Cisternas, & Rivas, 2017b; Grabiec et al., 2017; Han et al., 2017).

2.8.3. pH measurement The influence of pH on MICP is indisputably important because it affects microbial

activity or bacterial growth, urease activity, and CaCO3 precipitation (Kim et al., 2018). The increase in pH is often due to the hydroxide ions (OH−) generated during ureolysis which causes change to the pH medium or solution (DeJong, Mortensen, Martinez, & Nelson, 2010).

The decomposition of urea by urease that releases NH3(g) and CO2(g) and the dissolution of

NH3(g) in the urea-CaCl2 leads to a variation of pH during ureolysis process (Li et al., 2013). Kim et al., (2018) recently reported that during MICP process, certain bacteria such as Staphylococcus saprophyticus is not sensitive to changes in pH of cementation solution and

only fairly affected its CaCO3 precipitation (20% decrement). Unlike S. pasteurii which is significantly influenced by changes in pH (60% decrement). Hammes and Verstraete, (2002) and Stocks-Fischer et al., (1999) were among the earliest scholars to explain how pH affects

the metabolism pathways of MICP for CaCO3 precipitation. pH has a critical impact on biomass growth and enzymatic activity, such as when it is higher than pH 10 but may do not

inhibit CaCO3 precipitation to occur if sufficient calcium ions are provided (Cuzman et al., 2015).

Hence, it is imperative to study or monitor pH performance of ureolytic bacteria during MICP experiments. Typically, a pH meter is used to measure the acidity or alkalinity of the bacterial culture or suspension with the number of hydrogen ions (H+) or hydroxyl ions (OH-) (Omoregie et al., 2019). The pH of ureolytic bacterial cells in the nutrient medium can be measured during MCP experiment at a regular time interval for proper physiological characterization of its performance during cultivation (Achal & Pan, 2014). The pH meter is usually calibrated with commercial pH standard before the pH measurement on samples is performed, after which the pH sensor is flushed with enormous water before the pH of next samples are determined (Soon, 2013). The MICP studies also analyse effluents collected from variously sampling sources or draining outlets during the MICP process for pH measurement (Lee et al., 2012, 2019a; Omoregie et al., 2019). Although, collected effluent samples are also subjected to cell viability or biomass and ammonium concentrations test (Cheng & Cord- Ruwisch, 2013). Changes in pH of effluent samples during or after MICP treatment provide a good indication of the state of the MICP process, (Nayanthara et al., 2019; Soon, 2013). Thus,

indicating the presence or reduction of urease activity and CaCO3 crystals.

2.8.4. CaCO3 content

The CaCO3 content is one of the most undeviating manifestations of MICP process and an important indicator which demonstrates the performance of the treatment technique performed

(Al-Salloum, Hadi, et al., 2017). The contents of CaCO3 precipitated within the soil matrix has

a substantial effect on the mechanical properties of the treated soil. Typically, CaCO3 content is often used to evaluate whether sand columns are well solidified by studying the distribution

of CaCO3 content after curing is completed (Jiang & Soga, 2017; Sun, Miao, & Chen, 2019).

Hence, measuring the CaCO3 content in the soil specimens after MICP treatment is often performed by many researchers. Also, in-vitro biomineralization test for CaCO3 content is

often carried out in glassware (e.g. flask) to evaluate the CaCO3 capacities or urease activity of the selected MICP microorganisms (Arias et al., 2017b; Dhami et al., 2017; Omoregie et al., 2019). Flasks which containing a certain concentration of cementation fluid are inoculated with

bacterial inoculum and incubated at a certain temperature. For evaluation of CaCO3 content from treated soil specimen which is often carried out after compressive strength test, conventional gravimetric acid washing method is commonly used because of its easy operation and analysis (Choi et al., 2017; Mahawish et al., 2019).

The differences between the two weights of the MICP-treated soil specimen is considered to be the weight of the carbonates that were precipitated (Rebata-Landa, 2007). Alternatively, ethylenediaminetetraacetic acid (EDTA) titration method is also used for determination of

CaCO3 content from biocemented soil specimens (Chu, Stabnikov, & Ivanov, 2012; Stocks- Fischer et al., 1999).

2.8.5. Compressive strength test The compressive strength test is commonly performed to verify MICP on treated soil specimens under desired conditions (Cheng, Cord-Ruwisch, & Shahin, 2013). Commonly, UCS test which is also known as a uniaxial compression test. The UCS test is widely used in geotechnical engineering to measure the axial load of stress applied to the subjected tested specimen along a longitudinal axis. First, the consolidated soils are carefully dismantled from the columns before the UCS test is conducted under controlled conditions (Wen et al., 2019). The UCS test is also frequently conducted to compare the performance of compressive strength for the treated soil samples for CaCO3 content (Cheng et al., 2014). Typically, the tested soil samples (i.e. diameter to height ratios of between 1:1.5 and 1:2) are subjected to axial load (constant rate of 1.0 mm.min-1) (Cheng & Cord-Ruwisch, 2014; Cheng et al., 2013; Cheng, Shahin, & Cord-Ruwisch, 2017; Mahawish et al., 2019).

In some cases, before the UCS test, the consolidated specimens are dried in an oven (i.e. 105oC) for at least 24 h (Chuangzhou, Jian, Liang, & Shifan, 2019). However, in most cases, the soil specimen will be allowed to air-dry or cure (i.e. 14 to 28 days) before dismantling it from soil columns (Omoregie et al., 2018). This test is often performed under laboratory- condition and the equipment is quite costly to procure. However, there are other inexpensive ways to measure the strength of biocemented soils. Surface strength or local strength measurements are also used to obtain desired compressive strength results of consolidated soil specimens. This can be obtained by using soil pocket penetrometer, rebound hammer (Schmidt rebound hammer) or needle penetrometer tests (Cheng & Cord-Ruwisch, 2014; Gowthaman, et al., 2019; Omoregie et al., 2017). These devices can be highly useful for MICP field-scale assessments of treated soil (Gowthaman, Iki, et al., 2019).

2.8.6. Microstructural analyses

The SEM and XRD both provide fervent qualitative and quantitative indication of CaCO3 precipitation after MICP process. While SEM is used in MICP investigations for virtual visualization of carbonate crystals in association to microbial cells incorporated in the system, XRD serves as a chemical assay which quantitatively determines the chemical composition information present in the tested specimen (Al-Salloum, Hadi, et al., 2017). These microstructural analyses are both considered non-destructive testing methods which could be used to analyse the consolidated soil materials. The mechanical behaviour of geomaterial is highly influenced by its microstructure (Jiang & Soga, 2017). Hence, microstructural investigation allows a better understanding of the crystal formation (i.e. shapes) and bonding

behaviour between the sand particles and CaCO3 crystals (Cheng et al., 2013; Cheng et al., 2014). The SEM equipment also has attached to its EDX feature which can provide quantifiable information from specimens about its elemental compositions through the use of X-ray analyses which uses electrons to analyse the surface of the specimen.

2.8.7. Other testing procedures There are other numerous analyses which are also being used to generate essential information on the performance of MICP on treated soil specimens. These other testing procedures include Fourier-transform infrared spectroscopy analysis (Xu et al., 2017); plasticity index characterization (Osinubi, Eberemu, Gadzama, & Ijimdiya, 2019); cone penetration test (M. G. Gomez et al., 2015); porosity and permeability tests (Mahawish et al., 2019; Wen et al., 2019); water absorption test (Porter, Dhami, & Mukherjee, 2017), triaxial compression test (Cheng et al., 2013); and X-ray fluorescence analysis (Nayanthara et al., 2019). These procedures are also being used to elucidate and determine MICP potential usefulness for other applications.

2.9. Potential applications of MICP Numerous studies in the literature have shown the potential of using MICP technology to resolve biotechnological, engineering and environmental challenges. A detailed discussion of past and recent studies which cover slope stabilization, bioremediation of heavy metal, restoration and production of structural materials, mitigation of soil liquefaction, erosion and seepage control and other applications (CO2 sequestration, enhanced oil recovery, polychlorinated biphenyls removal, wastewater treatment and utilization of industrial by- products) are momentarily discussed in the ensuing paragraphs.

2.9.1. Slope stabilization Slope soil stabilization is getting increased attention as slopes are frequently associated with transportation systems (Bao et al., 2017; Jiang & Soga, 2017; Salifu et al., 2016). Slope failure remains a geotechnical challenge in most coastal and estuarine regions due to constant exposure to tidal currents (Salifu et al., 2016). The surface treatment is important in promoting the cover condition of slopes by achieving aggregate stability and infiltration control (Gowthaman, Iki, et al., 2019). Erosion and heavy rainfall are tremendously responsible for most soil landslides due to pressure and seepage forces (Bao et al., 2017; Muntohar & Liao, 2010). Surface runoff depth and surface soil loss rate get increased with the increase of rainfall intensity (Dai et al., 2018). Also, soil texture, slope topography and cover condition influents the runoff production of a slope because they are unsaturated (Gowthaman, et al., 2019; Zhang et al., 2018). There are no known suitable stabilization approaches available in the literature that could prevent slope failure from occurring with minimal implications (Salifu et al., 2016). Failure of embankment slopes causes damage to transportation systems and often require high cost for maintenance (Bao et al., 2017; Gowthaman et al., 2019). To promote slope enforcement or prevention measure, some approaches are commonly utilized which includes the addition of foreign soils as reinforcement (Liu & Li, 2003); chemical cementation (i.e. fly ash, lime or cement) (Liu et al., 2011); biogenic methods (Burbank et al., 2013) and physical installation of membrane structures (i.e. geotextiles, wire meshes, cable nets) (Kumar, 2010).

Regrettably, there are limitations to some of these conventional methods such as requiring high energy and operational cost, utilization of heavy machinery which may disturb existing soil bodies and high viscosity of chemical stabilizers which are also regarded as ecologically unfriendly (Salifu et al., 2016; van Paassen, 2009). Since MICP is a non-destructive and less hazardous when compared to conventional soil improvement methods, it has received enormous attention from researchers as a potential candidate for slope surface stabilization (Gowthaman et al., 2019). Salifu et al., (2016) performed a preliminary investigation by applying MICP on selected sandy slope specimens in a perspex box container (having equal sides of 200 mm). The MICP was evaluated as a remedial measure to treat steep slope (53°) by using S. pasteurii ATCC 11859 (OD of 1.0) and cementation fluid (0.7 M of CaCl2 and 0.7 M of urea). The MICP treatment with higher cementation concentration results in the enlargement of soil particles and the change of pore shapes, which are attributable to the formation of more calcite precipitation as cementitious bonds (Jiang & Soga, 2017). Salifu et al., (2016) reported MICP process produced 120 kg calcite per m3 of soil which resulted in filling 9.9% of soil pore space. Also, the biocalcification withstood 470 kPa compressive stress, showing improved slope stability. Although they also reported not having heterogeneous distribution or penetration of calcite in their field trial experiments and suggested it could be due to unconsidered factors that occur during the scaling-up process. Salifu et al., (2016) suggested that their MICP treatment technique would be suitable for the embankment of slope having a relatively shallow penetration treatment (10–20 cm).

Recently, Gowthaman et al., (2019b) carried out a preliminary slope soil stabilization test under small-scale laboratory-condition to determine the feasibility of using locally isolated bacteria (Psychrobacillus sp) from Japan and low-grade chemicals. MICP treatment under various temperature and injection sources were performed in a syringe (diameter of 25.3 mm and height of 139.6 mm) which contained 45 g of soil for the slope stabilization test. Their studies showed that the native bacteria had optimum treatment conditions for preventing soil slope when grown at 30oC, 0.5 M of cementation solution are used for treatment and bacterial cells are injected only at the beginning of the treatment period. More so, the compressive strength values for soils treated with low-grade chemicals at 30oC was 0.82 MPa, while those treated with the pure-grade chemical was lesser (0.417 MPa). Gowthaman et al., (2019b) stated that another reason MICP treatment was successful in their test favoured the type of soil they used.

Since slope soil was well graded with coarse material and gravel content (20 to 25%), it permitted the necessary CaCO3 content needed at the contact zone layers of the soil particles to enhance solidification. Their study did not explain the matrix supporting mechanism which allowed the low-grade chemicals to outperform the pure-grade chemical but they showed that this was due to higher CaCO3 crystal contents. Gowthaman et al., (2019a) also demonstrated that MICP could be implemented as a sloping soil stabilizing method for cold subarctic regions. They highlighted that MICP is not often applied in this region because ureolysis process tends to decline due to lower temperature which therefore results in insufficient CaCO3 precipitation. Hence used locally isolated ureolytic bacterium (Lysinibacillus xylanilyticus) from native soils and cementation fluid (1 M of CaCl2 and 1 M of urea) to treat slope soil model (13 × 10 × 10 cm). Using an injection rate of 8 to 10 mL.min-1, they bioaugmented the indigenous bacteria for two cycles before injecting 14 treatment cycles of cementation fluid for 14 days. The augmentation of indigenous bacteria provides an important environmental benefit during MICP process because they eliminate non-native microbial pollution into natural soil ecosystems and are acclimatized to the local environment, hence may promote necessary enzymatic activities and biomass production (Gomez et al., 2016; Gowthaman, 2019). Their result showed that L. xylanilyticus showed significant urease enzyme at temperatures 15–25 °C and was most suitable for MICP treatment in the cold subarctic region. The treatment process successfully cemented (over 80%) the slope specimen, had a compressive strength of 2–8 MPa and a stiff layer of around 3 to 4 cm along the entire surface was achieved (Fig 2.17).

(A) (B)

Fig 2.17: Slope stabilization model test: (a) Schematic diagram of the experimental arrangement of slope model and (B) profile of the cemented slope model (Gowthaman et al., 2019). Reprinted with permission from Springer Nature.

2.9.2. Bioremediation of heavy metals Heavy metal pollution is a serious environmental issue that causes economic, human and ecological challenges (Hossen, Hamdan, & Rahman, 2015). Globally, heavy metals (i.e. lead, mercury, and copper) that have densities more than 5 g.cm−3 are regarded as highly toxic (He, Chen, Zhang, & Achal, 2019). They exist naturally in biota and abiotic components as essential nutrients for living organisms especially when present at low levels (Li et al., 2013; Zhang et al., 2019). Rapid industrialization and the human population has contributed tremendously to the increase of metals and metalloids accumulation in the environment (Tamayo-Figueroa, Castillo, & Brandão, 2019). Unwanted release of heavy metals occurs as industrial waste, landfill, fertilizer and pesticides, burning of fossil fuel and municipal waste treatment (Li et al., 2013). In some cases, improper disposal of these heavy metals into water bodies affects and contaminates the food web of marine organisms (Hossen et al., 2015). Also, dams and reservoirs which are mostly built for irritation, water supply or electrical generation are causative agents of heavy metals accumulation which lead to unwanted fatality and diseases (Sim et al., 2016).

Previously, to control or treat the biomagnification of heavy metals, various conventional treatment methods (i.e. chemical precipitation, filtration, ion exchange, reverse osmosis and membrane technology) were used but regarded ineffective, ecologically unsustainable, too expensive because they required high chemical quantities for treatment (Arias et al., 2017a; Krishna & Philip, 2005). These remediation methods have become inefficient when heavy metals concentrations were less than 100 mg.L-1 (Arias et al., 2017a). Decontamination of soils polluted with heavy metals and different oxidation states may also result in increased toxicity levels, making many current bioremediation treatment techniques (e.g. phytoremediation) unsuitable (Zhu et al., 2016). Therefore, suitable alternative bioremediation techniques which are also cost-effective are highly needed (Zhang et al., 2019; Zhu & Dittrich, 2016). This attracted the attention of many researchers to explore using ureolysis-drive MICP process for heavy metal remediation due to its potential proficiency in the immobilization of these metals elements. The MICP technology was initially not considered as a suitable bioremediation method for removal of heavy metals but improved understanding of its process and mechanism has provided lucid scientific information for the development of new sustainable techniques to remediate heavy metals (Mugwar & Harbottle, 2016; Mwandira et al., 2019).

The reason ureolysis-driven MICP process is now considered an appropriate method to remediate heavy metal is that ureolytic bacteria have the large surface area and available anions on their cell surfaces which allows them to bind easily to vast heavy metals (Wong, 2015). Another advantage of using ureolysis process for heavy metals bioremediation is due to its ability to successfully immobilize these toxic metal through precipitation or co-precipitation

with CaCO3 irrespective of its concentrations (Kumari et al., 2016). Although there are challenges such as pH regulation or control of the system which affects the optimum performance of the MICP process on heavy metal (e.g. copper) remediation (Duarte-Nass et al., 2020). The same authors suggested this issue can be addressed by cultivating ureolytic bacterial without the presence of heavy metal to prevent growth inhibition and then mixing the biomass in a solution containing a high concentration of heavy metal. They suggested that implementing this two-step process would allow higher degree removal of heavy metals.

While many studies have successfully shown the removal of various heavy metals through MICP process, investigations by Achal et al., (2011) and Achal et al., (2012) provided essential guides on how to properly treat soils contaminated with unwanted metals. Although it is vital to note that studies on bioremediation of heavy metals (i.e. strontium and lead) in groundwater by MICP process have been previously performed (Fujita et al., 2004). Achal et al., (2011) showed that Kocuria flava CR1 could remove copper (95 to 97%) from contaminated samples. After determining the influence of initial pH (6.0 to 9.0), temperature (25 to 450C) and copper concentration (100 to 1000 mg.L−1) on copper bioremediation, copper remediation on soil samples were performed. Another study by Achal et al., (2012) demonstrated the versatility of the MICP process for bioremediation of heavy metal in soil contaminated with arsenic high concentration (500 mg.kg−1). For soil treatment, they used S. ginsengisoli CR5 (107 CFU.mL−1), a Calcifying arsenic resistant bacterium isolated from soil sample contaminated with arsenic. The bioremediation study was done on soil contaminated with arsenic (500 mg.kg−1) for 10 days at 30 ◦C. Their result showed (Fig. 2.18) that arsenic concentration in the exchangeable fraction of tested soil specimen significantly reduced from 25.85 to 0.88 mg kg−1. These two studies suggested that the ureolytic bacteria used in their investigations were successfully able to remediate heavy metals (copper and arsenic) due to their significant urease activities (472 U.mL-1 for K. flava CR1 and 412 U.mL−1 for S. ginsengisoli CR5). Also, heavy metal bioremediation was successful because MICP allows microorganisms to secrete metabolic products which then react with ions or compounds in the microenvironment, resulting in deposition of mineral particles (Achal et al., 2012).

These biominerals act as mineralogical trap or retention site for heavy metals from contaminated soils. Kang et al., (2014) investigated the removal of cadmium in aqueous solution using Lysinibacillus sphaericus CH-5 which was previously isolated from a mining region. The effect of cadmium on the bacterial cells were first determined before evaluating its bioremediation potential. MICP treatment by adding bacterial cells grown for 24 h into a -1 -1 -1 solution containing beef extract (3 g.L ), peptone (5 g.L ), urea (20 g.L ) and CdCl2.5H2O (2 g.L-1). After 48 h incubation at 30 °C, it was observed. Their data showed that with urease production of 2.41 μmol.min-1, L. sphaericus successfully removed 99.95% of cadmium from the aqueous solution. In another study reported by Kumari et al., (2014), they determined the feasibility to use Bacillus cereus YR5 of previously isolated from river sample for treatment of artificial chromium.

Kumari et al., (2014) subjected soil (5 kg) which had 1.8 mg.kg-1 background of content

in a plantation pot containing, then later added bacterial cultures (500 mL) and K2Cr2O7 (100 mg). After 4 weeks, the five-stage Tessier sequential extraction method was used to determine the bioremediation efficiency of B. cereus YR5. They noticed that the exchangeable chromium was reduced from 100 to 1.86 mg.kg-1 compared to 39.3 mg.kg-1 in untreated soil sample which was attributed to abiotic matter.

Fig.2.18: Arsenic concentration in soil fractions for the control and bioremediated arsenic contaminated soil samples (Achal et al., 2012). Reprinted with permission from Elsevier.

2.9.3. Restoration and production of structural materials The importance of MICP in strengthening, improving the durability or repairing concrete and mortar structures have been fervently investigated by several researchers. While many studies on utilizing a variety of ureolytic bacteria for structural repair, only a few investigations on that report the potential of fungi and co-cultures (fungi-bacteria) for CaCO3 precipitation have been conducted (Xu et al., 2020). Structural material such as brick masonry constitutes a significant part of construction material found in a historic building (Raut, Sarode, & Lele, 2014). This historical structure which holds essential cultural and economic values to numerous societies around the world requires maintenance due to deterioration. Concrete buildings or mortar monuments are usually exposed to deterioration because of weathering cycles (i.e. climatic, environmental, and substrate conditions) and in some cases face unrepairable damages. Globally, the cost for infrastructure rehabilitation is overwhelming and often due to the presence of chloride in these structures, hence leading to an increase in the severity of corrosion attacks and deterioration mechanism (Penttala, 2009; Tahri et al., 2018). Mortars based on organic binders (i.e. epoxy resin or polyester) or those based on inorganic binders (i.e. Portland cement) are typically used to treat these deteriorated structures but provide insufficient consolidation (Rodriguez-Navarro et al., 2003; Tahri et al., 2018). Unfortunately, these materials are regarded as unsuitable for the environment due to their association with toxic side effects and production of GHG emissions.

The MICP technology has recently been seen as a suitable alternative which may be used to repair decaying monumental and concrete structures. Application of MICP in cement-based materials has become substantially popular in the recent decade (Joshi et al., 2018a). This could be attributed to the ease of MICP treatment and its efficiency in repairing damages on concrete structures which may have been caused by environmental factors or human influence. Adolphe et al., (1990) were among the first group of researchers to harness the potential of MICP for the protection of ornamental stone. They showed that MICP techniques had the potential to resolve practical problems. Their patent on this technology which saw the rise of numerous biomaterial or bioconcrete studies showed the avid potential of MICP technology. Another

early MICP study was done by Chunxiang et al., (2009), they proved that CaCO3 precipitation through ureolysis process could provide protection and consolidation for concrete surface

layers from corrosion. Their laboratory-based experiments showed that CaCO3 induced by S. pasteurii was able to enhance the surface permeability resistance to acid attack.

Xu et al., (2018) demonstrated that a Bacillus sphaericus had the potential of improving concrete cracks as long optimum conditions (i.e. 108 cell.mL-1, alkaline pH, 2 M of urea and 0.9 M of Ca2+). They immersed mortar cubes (50 x 50 × 50 mm) prepared with OPC and sand into growth nutrient containing bacterial suspension before oven (40 °C) drying it. Upon completion of the MICP treatment, the self-healing efficiency of the bioconcrete specimen was confirmed based on visual inspection on crack closure and SEM analysis. Manzur et al., (2019) recently showed S. pasteurii could be used for repair of brick aggregate concrete specimens. Their work was initiated by incorporating bacterial cells into the aggregate specimen which had a mix ratio of 1:1.5:3 (corresponding to cement: fine aggregates: coarse aggregates, respectively). The samples were submerged into a cementation solution containing (140 mL of

CaCl2.2H2O and 400 mL of urea) for 21 days. Findings from the water absorption test (WAT) suggested that MICP was able to decrease water absorption ranging from 6 to 18%. Durability tests such as rapid migration test (RMT) and rapid chloride penetration test (RCPT) also showed a reduction in the rate of penetration (14.3 to 42.9%) and amount of electrical charges passed (7.0 to 27.3%), respectively.

Several studies have demonstrated that biobricks has the potential to compete commercially with conventional bricks from the perspective of GHG emissions. In a study by Iezzi et al., (2019), it was reported biobricks must cost between USD 0.03 to USD 0.07 for GHG emission to be lowered and to be commercially accepted as a cost-effective product. This may be achieved through mitigating wastewater treatment, decreasing resource consumption and lowering the cost of treatment reagents. Procedures to improve the mechanical properties of concrete structures could be economized by the spraying of bacterial cultures and cementation solution after preparing the concrete specimens (Tripathi, Anand, Goyal, & Reddy, 2019). Kalhori and Bagherpour, (2017) reported that MICP process improved the properties of specimens subjected to shotcrete crack healing. Visual observation from their experiments displayed significant healing in treated concrete samples which previously had cracks, thus enhancing the durability of shotcrete structures (Fig. 2. 19). Prior to starting the -1 -1 spraying process solution containing (20 g.L urea and 49 g.L of CaCl2.2H2O) and Bacillus subtilis PTCC 1254 (2.2 x 106 cells.cm-3), wooden panels (width of 610 mm, length of 610 mm and depth of 100 mm) were prepared and mixed with the shotcrete specimen. To examine the self-healing properties in the shotcrete specimen, artificial cracks were made before curing the samples.

Fig. 2.19: Digital images showing repairs of cracks in shotcrete specimens using MICP process after 28 days of curing period (Kalhori & Bagherpour, 2017). Reprinted with permission from Elsevier.

Joshi et al., (2018b) also investigated the influence of nutrient ingredients in solutions used to improve structural properties of concrete specimens. The ureolytic bacterial cells were added to the specimens during the casting stage or by spray treatment after casting mixes. Their findings also indicated repair capabilities of MICP on concrete specimens similar to those reported by other researchers. They recommended that to obtain optimum performance of MICP process on concrete and mortar structures, bacterial cells admixed treatment method should be done on newly produced casted specimens (i.e. concrete), while bacterial spray treatment method is used for repair procedure. Raut et al., (2014) revealed that MICP treatment for biobrick production was reduced when they used optimized growth media resulted in 13.3 % cost savings when compared to a solution containing nutrient broth. The researchers used conventional red bricks (200 mm x 100 mm x 70 mm) which contained 80% of clay, 15% of ash and 5% of rice husk for the biocalcification experiment. The prepared bricks were immersed in a solution containing S. pasteurii culture suspension (1.5 OD), growth medium and cementation reagents (2% of urea, 0.3% of CaCl2) and cured for 28 days. Their findings showed that compressive strength and WAT of the biobrick treated in the cementation solution containing the optimized growth media were 7.54 N.m-2 and 48.9%, respectively. In another study by Bernardi et al., (2014), MICP process aided the production of biobrick which resulted in compressive strength of 1 MPa to 2 MPa. Further studies on practical cost consideration for concrete repair by Lambert and Randall (2019) showed that this could be done by utilizing 31 L of human urine (an alternative to calcium and urea sources) via MICP. Their treated concrete specimen had a compressive strength of 2.7 MPa. Lately, Abdulkareem et al., (2019) used Bacillus subtilis to evaluate the effects of various atmospheric curing condition on compressive strengths of treated cement mortar samples. The ureolytic bacterial cells (109cells.mL-1) were added to concrete mortar through direct mixing. Their treated mortar specimens had compressive strength increments after curing for 7 days (53.7% and 120.6%) and 28 days (44.9% and 130.6%), respectively. Work from this research group also revealed that 40oC and 95% relative humidity had a remarkable influence on the compressive strength of the treated mortar specimens. These conditions resulted in a 133% increase of the strength measured, but direct exposure to sunlight led to a detrimental compressive strength value.

2.9.4. Mitigation of soil liquefaction Mitigating soil liquefaction has become an emerging area for MICP researchers (Osinubi et al., 2020). Early evidence for the phenomenon of strength loss and increment in pore pressure after avid flow slides from vibration near a railway bridge at Weesp was recognized and reported by a group of Dutch engineers in 1918 (Jefferies & Been, 2015). However, Hazen (1920) was the first to use the term “Liquefied” to describe the behaviour of sandy soil after the failure of Calaveras Dam. The intricacies surrounding soil liquefaction makes it a serious issue geotechnical engineers ponder on (Muhammad, 2013). Liquefaction of granular soils happens when there are vibration or water pressures within the soils that lead to soil particles losing contact with one another and is often due to seismic waves passage through saturated sandy soils which are very loose (Shelley, Mussio, Rodríguez, & Chang, 2015). This condition regrettably acts like liquefied granular soil, making it unable to withstand the applied weight. Static or dynamic loading in these saturated sandy soils results in damages to existing structures or building foundations (Bao, Ye, Ye, & Zhang, 2016; Shivaprakash & Dinesh, 2017).

Soil liquefaction can also lead to the destruction of historic or prodigious structures, casualties to human life and economic losses (Bao et al., 2019). Soil liquefaction is related to earthquake and occurred in places such as New Zealand, China, Japan and Indonesia that are susceptible to various seismic hazards. For example, 144 people were killed in 1964 due to the earthquake that occurred in Niigata (Japan), resulting in more than $1 billion in damage (Jefferies & Been, 2015). In 1983, the desertion of Nerlerk (Canada) artificial island occurred after more than $100 million was spent on its construction due to soil liquefaction. Most recently, in 2018, an estimate of 5000 people succumbed to their death under debris in Balaroa and Petobo regions of Indonesia. The environmental issue arising from soil liquefaction motivated numerous researchers on developing methods (i.e. grouting with chemical solutions, foundation densification, gravel columns, foundation replacement and dewatering) for suitable resistance and control measures (Bao et al., 2019). Most soil treatment on soil liquefaction happens on sites where only important infrastructures (i.e. seaports and airports) are constructed (Chu et al., 2015). The need to resolve liquefied soils is problematic because of the high cost required for repair, environmental pollution and risk of damaging adjacent buildings when not handled properly (Bao et al., 2019).

Chemical grouting methods (i.e. cement and silicate) are commonly used for mitigate liquefied soil without the need for soil compartment and reduce risks of earthquakes but not preferable for large soil volume because it will require high energy and high cost (Chu, Varaksin, Klotz, & Mengé, 2009; Karol, 2003; van Paassen, 2011). Besides, it is often difficult to perform ground mitigation of liquefaction because the scale of the ground to be treated is normally vast, and too expensive to be implemented (Chu et al., 2009). Thus, new cost- effective and environment-friendly treatment methods for liquefaction mitigation must be developed so prevention or control can be performed especially in regions with vast surface areas (Chu et al., 2015; Huang & Wen, 2015). On this aspect, MICP has shown vital usage for ground treatment of its significant static strength capacity and ability to increase shear wave velocity of granular soils (Montoya & DeJong, 2015; van Paassen, 2009; Whiffin, 2004). Biogrouting is a recent innovative MICP technique which can be utilized in the fields of geotechnical engineering for ground mitigation of liquefaction. Just like many other MICP treatment techniques, biogrout is developed on the same basis (MICP process) and performed mainly by ureolytic bacterial cells to improve engineering properties of soils (Kumari & Xiang, 2019).

Van Paassen et al., (2009) were among the earliest researchers to investigate the usefulness of MICP biogrouting for ground stabilization under conditions and techniques which may be employed for in-situ practice. Van Paassen et al., (2009) experimented on large scale concrete box (8 m x 5.6 m x 2.5 m) which was filled with sand (dry density of 1560 kg.m-3) and injection (flow rate of 1 m3.h-1) was initiated. Their findings showed that the compressive strength (0–

12 MPa) and CaCO3 content (0.8–24%) varied throughout the column but interestingly, they noticed no bioclogging formation at the injection points. This may have been due to the use of higher flow velocity during treatment, which therefore minimized urease activity and formation

of CaCO3 from occurring at the entry points of injections. Given the success of the preceding investigation, van Paassen, (2011) reported the first field-scale biogrout experiment which was carried out in the Netherlands (collaboration with Deltares and Delft University of Technology). van Paassen, (2011) indicated that a soil volume of 1.000 m3 was treated using the MICP procedure (depth ranging from 3 to 20 m below the surface). The diluted bacterial suspension (200 m3) and cementation solution (300–600 m3) were injected (6 injection wells) into the boreholes. Results on electrical conductivity (apparent resistivity from 120-2 Ωm) and

CaCO3 content (6%) from this preliminary study showed good indication of MICP treatment.

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Montoya et al., (2012) previously showed that bio-mediated soil improvement via MICP was able to demonstrate improved resistance to pore pressure generation and reduce loose sand susceptibility subjected to seismic loading. Their team reported a significant reduction (50%) of excess pore pressures beneath the structure when compared to the untreated soil specimen. Lin et al., (2016) later presented a new alternative ground stabilization pile method to the biogrouted pervious concrete pile. Their work showed that by using an improved MICP

biogrout approach, the common bioclogging and heterogeneous CaCO3 distribution reported in several researchers was not encountered in this current study. Lin et al., (2016) investigated the possibility of enhancing the axial compression response of pervious concrete via MICP process (Fig. 2.20). Results from their experiment showed that biogrouting aided in improving the shaft resistance along the length of the pile from 56% to 90% of applied load (from non-

grouted pile to biogrouted pile) and CaCO3 crystals were seen on the shaft (Fig. 2.21).

Fig. 2.20: Instrumentation, MICP biogrouting and compression loading setup (Lin et al., 2016). Reprinted with permission from the American Society of Civil Engineers.

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Fig. 2.21: Digital image showing pile specimen subjected to MICP treatment for ground Improvement of soil (Lin et al., 2016). Reprinted with permission from the American Society of Civil Engineers

2.9.5. Erosion and seepage control The MICP has also been studied for erosion and seepage control by several MICP research groups. Since MICP has the feasibility to enhance soil mechanical properties, it may, therefore, be able to mitigate or prevent loss of granular soils. Recently, studies which subject MICP- treated soils to wind tunnel conditions at different intervals for determination of loss of mass due has shown impressive results (Osinubi et al., 2020). Wind erosion is an environmental factor that causes suspension of soil particles and desertification especially in agricultural regions (Han et al., 2007; Maleki et al., 2016). Wind erosion occurs in humid climates but it is more predominant in arid and semiarid areas, resulting in the destruction of agroforestry and dust storms (Maleki et al., 2016; Wang et al., 2018). Soil stabilizers (i.e. petroleum mulches and polymeric materials) are often utilized as surface or subsurface layer reinforcement to control wind erosion but have an environmental impact (He, Cai, & Tang, 2008).

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Seepage control is also a common construction process for various engineering infrastructure projects especially underground constructions (Yang et al., 2019). Earth dam is one of the mostly constructed hydraulic infrastructures but has drawn attention from the public on its safe usage due to frequent and unpredictable extreme weather conditions (Jiang, Soga, & Dawoud, 2014). Interestingly, internal erosion has been regarded as the third most important mode for earth dam failure of earth dam incidents after overtopping and external erosion (Jiang & Soga, 2017). Polymers and chemicals are often used to provide waterproof but some of these materials are deemed unsuitable due to their toxicity and environmental unfriendliness (Yang et al., 2019). Hence, MICP has rather been gained attention in recent years as a potential alternative which could address this problem. Maleki et al., (2016) investigated the use of MICP technique to minimize wind erosion risk of sandy soil in a wind tunnel (0.25 m x 9 0.25 x m 9 4 m) with S. pasteurii (2.2 mM urea.min-1 of urease activity and 1.5 g dry weight.L-1 of biomass cells) in which wind velocities (10 to 55 km.h-1) were applied. The soil columns which were subjected to MICP treatment were placed on a tray (0.03 m x 0.15 m x 0.15 m) as shown in Fig. 2.22. Their findings showed significant improvements in the penetration resistance of the treated soil samples and compressive strength recorded was 56 kPa. Wang et al., (2018) demonstrated through small-scale laboratory experiments that MICP could control wind erosion by improving the resistance and strength of the treated soil via spraying method. Their result showed and reduction of erosion rate for treated soils result in 90%. Tian et al., (2018) reported having UCS strength of 3.2 MPa and reduced permeability coefficient (1.08 × 10-3) on their aeolian sandy soil which was subjected to MICP treatment despite wind velocity risk (5.73 m.s-1), MICP process was able to lead to successful erosion control. Jiang et al.,(2014) utilized MICP treatment via a downward seepage flow and incremental flow rates. They reported that after MICP treatment, the critical gradient and critical shear stress had 236% and 81% increase, respectively. Besides, they also noticed a steady increase in erosion correlated with increasing with incremental flow rates. Jiang and Soga, (2017) recently explored MICP prospective applicability for internal erosion control in a mixture of granular soils (gravel and sand) using a large one-dimensional column test apparatus. The result from this research group showed that MICP treatment was successful in reducing the cumulative erosion weight, erosion rate and axial strain when compared with the non-treated soil. The magnitudes of hydraulic conductivity for all treated soil samples before the erosion process fall into a range from 5·5 x -5 -3 -1 10 to 8·0 x 10 m.s . They also noticed CaCO3 contents progressively increased with MICP treatment irrespective of hydraulic pressure.

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Fig. 2.22: Treated dunes with bacterial cells and cementation reagent for penetration resistance experiment (Maleki et al., 2016). Reprinted with permission from Springer Nature.

2.9.6. Wastewater treatment Water is an essential commodity which is required for numerous activities and survival of human and other living organisms, however high population growth increases the demand for good quality water (Ezechi, Affam, & Muda, 2020a). Most drinking water is obtained from water surfaces (i.e. dams, lakes and canals), however, freshwater availability has become a serious global problem especially in developing countries (Jonnalagadda & Mhere, 2001). This is directly attributed to the increase of human urbanization and industrial development. Continuous discharge of waste materials from domestic and industrial wastewater will regrettably affect freshwater availability. Wastewater produced in certain regions does not undergo full treatment due to the absence of efficient wastewater treatment facility (Edokpayi, Odiyo, & Durowoju, 2017). In some cases, regions in developed countries which have proper wastewater managerial system which meets international standards are unfortunately faced with ether poor design or maintenance problems that eventually affect the quality of the treated wastewater (Edokpayi et al., 2017).

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Improper disposals and treatments of solid wastes are known to pollute the water bodies. Leachate formations occurs due to the increasing amount of water contamination by landfill waste material and improper control of leachate migration would continue to affect water quality (Affam & Ezechi, 2020). If these waste are not properly treated before being disembarked onto environmental water, it may lead to more water pollution and affect aquatic life present. The high expense for wastewater treatment using conventional methods (i.e. membrane processes, adsorption processes, ion exchange processes, and biological processes) has been a concern for many stakeholders and the constraints associated with their treatments demands the need for alternative pollutant removal methods (Ezechi, Affam, & Muda, 2020b). New biotechnological technology has made it possible to recycle wastewater and enhance the quality of water (i.e. seawater and freshwater) (Arias et al., 2017a). It is important to introduce new sustainable wastewater treatment methods which are cost-effective and can be utilized commercially. Some studies in the literature have shown the potential application of MICP on the removal of secondary ions present as a contaminant in wastewater (Arias et al., 2017b; Hammes, Seka, De Knijf, & Verstraete, 2003; Işik et al., 2010). Hammes et al., (2003) was the first research group to prove MICP technology could treat calcium-rich wastewater samples. Their preliminary investigation showed that urea degradation (ureolysis) process can be integrated into existing biological wastewater treatment system which could help remove calcium ions inexpensively. They reported removing 90% of soluble calcium at the end of their experiment by stimulating ureolytic microorganisms in the wastewater through sedimentation in the biocatalytic calcification reactor. Işik et al., (2010) also reported using MICP mechanism with biocatalytic calcification reactor for removal of calcium-rich mineral from wastewater samples. A recent study by Anitha et al., ( 2018) showed that Bacillus cereus could successful remove calcium mineral having 431.7 mg.L–1 of hardness during water treatment. Arias et al., (2017b) recently demonstrated that ureolysis process could act as biomineralizers of calcium and magnesium ions present in seawater samples. Their study showed precipitation of 95% soluble calcium and 8% magnesium at the end of the MICP treatment tests.

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2.9.7. Microbial enhanced oil recovery Microbial enhanced oil recovery (MEOR) represent the utilization of living microorganism to extract oil situated in reservoirs (Lazar, Petrisor, & Yen, 2007). Preferences for enhancing oil recovery (EOR) with methods which have optimal yield has been influenced by economic and environmental issues, thus MEOR has recently been selected as a suitable alternative (Jeong, Lee, & Lee, 2019). This is because the MEOR process can increase EOR capability through biotechnological technology. Previously when oils are trapped in capillary pores of the formation rock, EOR methods (i.e. combustion, steams, miscible displacement, caustic surfactant-polymers flooding) were used for recovery of oil (Lazar et al., 2007). However, since petroleum crises which occurred in 1973, traditional EOR methods became less profitable. In some cases, during oil recovery exploitation, water which was often flushed on long-term duration became major problems in crude oil production (Wu et al., 2017). Besides, the petroleum industry makes up one of the most hazardous wastes known on earth (petroleum sludge) and treatment has been a major challenge in recent decades because some of the various widely-known approaches (incineration, freeze/thaw, pyrolysis, ultrasonic irradiation and biodegradation) which are involved in the treatment of this unwanted waste material used are either unsuccessful, expensive or unsuitable (Johnson, Affam, Johnson, & Affam, 2018). Thus necessitating the dispersion of essential microorganisms in petroleum reservoirs which can selectively degrade or metabolize oil components because of a preferred choice. (Lazar et al., 2007). The use of microorganisms for EOR in the literature is widespread because the process is regarded as feasible over the traditional approach. This may be because bioproducts can be produced using a cheaper and easily degradable substrate which has also supported numerous successful laboratory experiments and pilot experiments (Jeong et al., 2019; Patel et al., 2015). Most MEOR techniques utilize microbes to produce biosurfactants, biopolymers, gases and solvents which can minimize the interfacial tensions (oil and water or oil and rock) (Sen, 2008). For in-situ MEOR trials, they often stimulate indigenous microbial populations or introduce selected microorganisms, inject by microbial nutrients and biocatalysts (Kantzas et al., 1992). Despite the reported potential of many MEOR approaches used in the oil industry for EOR, its field-scale application has not been very favourable due to the inconsistency of its success (Jeong et al., 2019). MICP approach for EOR has started gaining interest from researchers because of its high-permeability potential and plug vast zones. Using MICP treatment can help lead to a redirection of water flood in the reservoir and high- yield of EOR (Jason, Michael, & Nüsslein, 2006).

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Earliest work on MICP technology for application on EOR was reported by Ferris et al., (1996) and Kantzas et al., (1992). Their research groups were able to develop novel permeability reduction process using a microbial agent (S. pasteurii) which could aptly be applied for EOR from petroleum reservoirs or aquifers. A recent study carried out by Wu et al., ( 2017) allowed oil recovery improvement from 44 to 83% due do the reduction in permeability and increase in plugging capability.

2.9.8. CO2 sequestration

The CO2 sequestration using MICP process has recently gained numerous interest among

researchers. The increase in atmospheric CO2 concentrations has been widely considered as an environmental issue which is responsible for global warming (Yadav, Labhsetwar, Kotwal, & Rayalu, 2011). Without any doubt, human activities since global industrialization have caused

the rise in global atmospheric pollution. Recent data on atmospheric CO2 concentrations has superseded 400 ppm which unfortunately is the highest levels recorded in over 800,000 years (Ritchie & Roser, 2017). A special report by the Intergovernmental Panel on Climate Change, (2018) estimated that global warming may reach 1.5 °C between 2030 and 2052 if nothing is tremendously done to minimize the concentrations of atmospheric GHG. Thus, there is an

urgent need to reduce the release of atmospheric CO2 concentrations (Anbu et al., 2016). MICP

microorganisms have the potential capacity to notably sequester CO2 concentrations, hence has

recently attracted efforts from many researchers. The MICP through microbial CO2 fixation is an emerging and promising technology which is regarded as an economic and dependable means to mitigate GHG emissions (Rossi, Olguín, Diels, & De Philippis, 2015). In this method,

CO2 is converted often converted into carbonate minerals that can form different biominerals (Anbu et al., 2016). Okyay et al., (2016) were the first to show that bacterial consortia (Sporosarcina, Sphingobacterium, Stenotrophomonas, Acinetobacter, and Elizabethkingia genera) isolated from caves and travertines could sequester different amounts of CO2. Recently, Liu et al., (2020) demonstrated for the first time that desert soil microbes were

directly involved in CO2 fixation via MICP. They studied the soil inorganic carbon formed by 13 the microbial population and tracked the profile of atmospheric CO2. Their result interestingly

disclosed that during atmospheric CO2 was assimilated in soil inorganic carbon via MICP process and microbial genes essentially determined the metabolic pathways required to induce biominerals formations produced by the desert soil MICP microorganisms.

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2.9.9. Removal of polychlorinated biphenyls Removal of polychlorinated biphenyls (PCBs) using MICP process has also started receiving noticeable research interest. The PCBs contaminant is released into the atmosphere via dumping of industrial wastes and landfills with PCB containing products and (Van Gerven, Geysen, & Vandecasteele, 2004). Using materials such as epoxy often result in resurfacing of the contaminant on the engineering structure after cleaning due to a rise in environmental temperature (Okwadha & Li, 2011). Phytoremediation especially plant-based remediation (i.e. Lespedeza cuneate, Picea glauca, and Phalaris arundinace) is a biotechnological approach which has been successfully used remediate PCBs from the environment because of it cost- effective and ecologically-friendly (Jing, Fusi, & Kjellerup, 2018). Unfortunately, phytoremediation requires long duration (i.e. 20 years) for complete remediation and extreme weather may lower the effectiveness of phytoremediation systems (Jing et al., 2018; Kaštánek et al., 1999). Hence. MICP technology has been seen as an alternative which can remediate PCBs-contaminated areas (Anbu et al., 2016). Okwadha and Li, ( 2011) determined if MICP process could serve as a suitable alternative to an epoxy-coating system. Their result showed that after MICP treatment, the deposition of CaCO3 precipitates acted as biosealant (encapsulate) on the surface of their concrete specimens (Fig. 2.23), thus resulting to reduced permeability.

Fig. 2.23: PCB-contaminated concrete cylindrical specimens subjected to MICP treatment (Okwadha and Li, 2011. Reprinted with permission from Elsevier.

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2.10. The main challenges affecting MICP technology

2.10.1. Economic feasibility As opposed to many soil improvement methods, MICP is still relatively too expensive for field-scale implementation (Mujah et al., 2017). The economic limitations which affect the MICP process need to be addressed (Anbu et al., 2016). For example, The cost of bacterial paste (US$1421.kg-1) with a mixture of nutrients (US$233.kg-1) for treatment on building materials (i.e. mortar) is too costly when compared to traditional binding agents (approx. US$1.kg-1) (De Muynck et al., 2010). Until now, most MICP researchers use laboratory-grade growth media (i.e. nutrient broth and yeast extract) for biomass production which impedes MICP field-scale application. Some studies have shown that the use of industrial by-products (i.e. dairy waste and lactose mother liquor) can serve as alternative nutritional sources and be suitable for field-scale implementation because they are rich in protein sources (Achal et al.,

2010; Cuzman, Richter, et al., 2015). The MICP cost is also influenced by reagents (e.g. CaCl2 and urea) required for soil treatments and specific treatment techniques used (Wang et al., 2017). There are not diverse information on the actual cost of various MICP treatments available in the literature (Burbank et al., 2013). However, few researchers have elaborated on the cost MICP and its influence on-field implementation. Ivanov and Chu, (2008) indicated that the raw material costs for chemical grouting (up to US$7.m-3) are cheaper than microbial grouting (up to US$9.m-3). De Muynck et al., ( 2010) indicated that the cost of MICP process for surface treatments of sculptured and degraded stone ranged from US$29.71.m-3 to US$51.67.m-3. Ivanov and Chu, (2008) further specified that procurement of cementation reagents for field-scale MICP treatment will cost US$5.m-3. DeJong et al., (2011) and Esnault- Filet et al., (2012) showed that it will require US$25.m-3 to US$500.m-3 for the total cost of MICP soil treatment which includes materials, installation and equipment. However, Cheng and Cord-Ruwisch, (2012) suggested that the material costs for MICP field-scale work may be minimized if resourceful cementation is being adopted. More so, the cost of MICP treatment may be further reduced if efficient or optimized treatment technique is implemented (Wang et al., 2017). Due to the price of MICP constituents, it will be difficult to compete with conventional soil solidification methods soley on economical basis (De Muynck et al., 2010).

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2.10.2. Treatment uniformity

Homogenous distribution of CaCO3 contents along the treated soil matrix from respective injection point remains a critical aspect of MICP process (Mujah et al., 2017). It is one of the main obstacles affecting MICP technology as a practical ground improvement method (Shahin

& Cheng, 2018). This is because the uniformity of CaCO3 precipitates significantly influences

the strength and stabilization of the treated soil specimens. Deprived transportation of CaCO3

corresponds to poor mechanical strength attained. Uneven distribution of CaCO3 precipitates is often attributed to bioclogging formation which typically happens during treatment at the injection area. This happens because when microorganism travels through the pore space of soils are likely to react with the cementation solution immediately upon injection or percolation (Ginn et al., 2002). When unwanted retention or attachment of bacterial cultures and the cementation reagents happens at injection points, it limits the amount of required solution to pass through the soil matrix (El Mountassir, Minto, van Paassen, Salifu, & Lunn, 2018; Mujah et al., 2017). If bioclogging formation is not minimized or prevented during MICP treatment, it would hinder sufficient CaCO3 content. This then leads to uneven distribution of CaCO3 content within the soil matrix which affects the shear strength and stiffness of the treated soil (Omoregie et al., 2018). It is well-known that MICP treatment injection and surface percolation

methods often leads to uneven distribution of CaCO3 content (Omoregie et al., 2017; Røyne et al., 2019). So many scholars have reported experience highly variable or inhomogeneous

CaCO3 content and compressive strengths of their treated samples (Cheng & Cord-Ruwisch, 2013; Dhami et al., 2016; Røyne et al., 2019; van Paassen, 2009; Whiffin, 2004). Large-scale treatment specimens are easily prone to bioclogging or inhomogeneous distribution of precipitated material contents especially is an ineffective injection strategy is used. DeJong et al., (2010) reported that for large-scale and field-scale MICP techniques, the most important issue to consider during treatments is the homogeneity and durability of soils. (Shahin & Cheng, 2018). Since fixation and distribution of bacterial cells is really crucial in achieving homogeneous CaCO3 precipitates within the sand column while preventing formation of bioclogging at treatment point, two-phase injection procedure can be used where first introducing the bacterial cells suspension into the column and immediately inject the cementation solution at a slow rate (Harkes et al., 2010). The use of a pre-formed active urease

material (bio-slurry) to enhance soil stabilization and homogenous CaCO3 precipitates have been attempted (Liang Cheng & Shahin, 2016). More so, using a one-phase low-pH injection method can resolve the bioclogging problem (Cheng et al., 2019).

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Recently, Cheng et al., (2019) recommended the use of a simple novel one-phase injection treatment method as opposed to the multiple-phase (i.e. two and three-phase injection strategies) which helped inhibit bioclogging formation and heterogeneous distribution of

CaCO3 content. They experimented on how different pH would influence the bioflocculation induced by CaCl2 when prepared in a mixture of bacterial cells (OD of 0.33 to 2.24) and

cementation solution (1 M of urea and CaCl2). Also, to determine the feasibility of one-phase injection of low-pH all-in-one solution biocementation method, bacterial cells (urease activity -1 of 1.25 to 10.0 U.mL ) and cementation solution (2 M urea and CaCl2) were placed in a soil column (internal diameter of 50 mm and length of 120 and 360 mm, respectively) which contained silica sand. The injection treatment was done from the bottom of the soil column with a constant flow rate of about 1 L.h-1. The solutions were injected every 24 h until full saturation was obtained then cured at room temperature (25 ±1o C) for 24 h. Their results indicated that by varying the pH values, the percentage of biomass coagulation decreased with a decrease in pH, while it increased with biomass concentration. Hence, lower pH (5.5) was suitable for MICP process as it could help minimize bioclogging formation during injection treatment. They also reported that this technique allowed the injection treatment to be controlled for up to 35 min without experiencing any bioclogging formation. This resulted in UCS up to 2.5 MPa after successful 6 treatments.

2.10.3. Production of ammonium Ammonium production during MICP process is one of the major problems limiting commercial implementation (Mujah et al., 2017). A high concentration of ammonium production is commonly produced during for the formation of calcite precipitation, which has a detrimental impact to human health, soil, groundwater and environment (van Paassen et al., 2010; Wang et al., 2017). Ammonium is a common inorganic source of nitrogen in the soil system, but if the high concentration is released to soils and left untreated in surface and subsurface region, it can lead be problematic to the soil (Lee, Kolbus, Yepez, & Gomez, 2019). Ammonia emission is considered in the European Union as one of the top important air pollutants (Ivanov et al., 2019). If ammonia exposure to water is more than 0.5 mg.L-1, it can cause harm when consumed (Mujah et al., 2017). The environmental issue concerning ammonium by-products has remained largely unaddressed (Lee et al., 2019).

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Although, several scholars have provided suggestion on how to minimize, control or prevent this toxic pollutant. van Paassen, (2009) suggested replacing ureolytic bacteria with denitrifying bacteria as a replace for biomineralization process. Unfortunately, denitrifying bacteria requires a longer duration for high biomass concentration, have low activity and lesser calcite precipitates when compared to ureolytic bacteria (Wang et al., 2017). Additionally, the presence of ammonium in building materials which were subjected to MICP treatment can be

converted (nitrified) into nitric acid by a microorganism which may react with CaCO3 to form soluble Ca(NO3)2 that can cause deterioration to the building materials (Ganendra et al., 2014). Mujah et al., (2017) recently recommended the ammonia-rich effluent be treated after soil biocementation and back-feeding the effluent as a fertilizer to the surrounding plants. Lee et al., (2019b) performed a preliminary investigation on the removal of total ammonium after soil biocementation by treating the effluent through post-treatment rinse injections with high pH

(9.0), high ionic strength solution (500 mM of CaCl2) for 103 days. Their treatment technique resulted in nearly 99.1% removal of ammonium.

Lee et al., (2019a) investigated the possibility of removing ammonium production after soil biocementation in 3.7 m3 long columns by applying 525 L of a high pH (10.) and high

ionic strength rinse solution (200 mM of CaCl2) for 700 min. The result showed that the treatment technique resulted in more than 97.9% removal of ammonium. Alternatively, Cheng et al., (2019) showed that by using a low pH (4.0) during MICP treatment, their technique resulted in more than 90% removal of ammonium. They also indicated that by controlling the biomass concentration, urease activity and initial pH of the cementation solution, the unwanted and harmful by-product (ammonia) was lowered. However, this process may lower the

precipitated CaCO3 minerals and result in weaker compressive strength. It will be interesting

for future studies to investigate the effect of using this ammonia removal method on CaCO3 precipitation and overall MICP performance. Ganendra et al., (2014) previously showed that

Ca(HCOO)2 could also serve as an alternative calcium source. Although their MICP process utilized methane-oxidizing bacteria (Methylocystis parvus), their study was the first to show

that Ca(HCOO)2 could successful induce CaCO3 polymorphs (predominantly vaterite).

Interestingly, Ganendra et al., (2014) reported that the use of Ca(HCOO)2 did not release ammonia gas when applied on structural materials. Hence it may serve as a suitable alternative to urea hydrolysis because of its reduced pollution risk and deterioration of building materials.

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2.10.4. Scale-up of ureolytic bacteria Producing a large volume of ureolytic bacterial cultures will make MICP economically challenging (Anbu et al., 2016). For MICP to move from laboratory-scale and field-scale trials to more practical implementation, it will require sufficient microbial cells (El Mountassir et al., 2018). Since numerous laboratory-scale experiments have provided adequate information on the way to enhance the optimum performance of ureolytic bacteria for urease production and

CaCO3 precipitation. To date, only a few studies have attempted cultivating MICP bacteria using modified or custom-built reactor systems under non-sterile conditions. The use of sterile equipment for field-scale MICP application is not practical due to the sheer size of the construction machinery (Omoregie et al., 2019). Hence, in this situation, it is not susceptible to use non-sterile growth condition for cultivation of ureolytic bacteria since the cells may out- compete other non-desired microorganisms when urea is added or ammonia production occurs (Graddy et al., 2018). One of the earliest studies on non-sterile production of ureolytic bacteria for the scale-up purpose was carried out by Whiffin (2004). The author showed that a custom- built fibreglass airlift pilot-scale reactor (120 L capacity) was able to grow the cells of S. pasteurii at 30oC under non-sterile conditions when placed on-site. Industrial-grade Vegemite acetate medium (13.5 g.L-1), urea (10 g.L-1) and initial pH medium 8.15 were used for the pilot- scale bacterial cultivation. Although lower urease activity and biomass production were obtained after cultivation, successful biocementation of soil occurred. Cheng and Cord- Ruwisch (2013) demonstrated that ureolytic microbial cells could be grown from activated sludge samples by using batch-scale chemostats (1 L capacity) at 28oC for 140 h under non- sterile conditions. Their result showed that optimum urease activity (60 µmoL.min-1) was attained when yeast extract-based medium (20 g.L-1), urea (0.17 M), ammonium sulphate (0.17 M) and initial pH medium 9.5 were used for cultivation.

Aoki et al. (2018) recently reported using a low-technology down-flow hanging sponge reactor (170 cm3 capacity) to cultivate ureolytic bacteria from samples collected from a reservoir tank. The non-sterile enrichment cultivation (130 days) occurred at 25oC using yeast extract-based medium which contained urea (0.17 M). The bioreactor operation period was divided into four phases (0 to 23 days for phase one; 24 to 57 days for phase two; 58 to 99 days for phase three; and 100 to 130 days for phase four) depending on the yeast extract concentrations in the supplied medium. Their results showed high urease activity (10 µmoL -1 -1 -1 urea hydrolyzed.min .mL ) and CaCO3 precipitates (92 ±7 mg.mL ).

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Findings from their SEM analyses demonstrated biofilm-forming capacity of the enriched ureolytic microorganisms in the custom-built reactor (Aoki et al., 2018). Der Star et al., (2009) previously showed that S. pasteurii could be mass-produced by using a large-volume (100 L) inoculum to scale-up the production in a 5m3 on-site bioreactor. They investigated the possibility of using MICP by denitrification process as an alternative to ureolysis process for soil ground improvement. Since cultivating ureolytic bacteria was costly and it resulted in ammonia production, MICP by denitrification process could remove nitrate to nitrogen gas without the formation of any by-product (Pham et al., 2017). However, Der Star et al., (2009) stated the main disadvantage of denitrification process when compared to ureolysis process

was its lower CaCO3 precipitation rate. Hence, the use of MICP by denitrification process for soil solidification is not adequate for in-situ applications (Tziviloglou et al., 2016). Therefore, there is a need to explore the influence of performing MICP experiments under non-sterile conditions and determine the consequence on ureolysis-driven MICP process. Most studies on scale-up of ureolytic bacterial species are done in reactors (1 to 5 L) under laboratory- conditions but for industrial applications of MICP to occur, more investigations on large cultivation in larger reactors should be performed (Zhu & Dittrich, 2016). Since the few studies of non-sterile cultivation of ureolytic bacteria for MICP application have demonstrated that in- situ scale-up using simple technology is possible.

2.11. Future implications For over two decades, MICP technology has shown ardent potential for various biotechnological and engineering purposes (i.e. erosion control, slope stabilization and heavy metal remediation). Its effectiveness has been well documented and widely proven under numerous laboratory-scale conditions. The research work on the MICP process from microscopic-scale to macroscopic-scale has shown vital progress (Wang et al., 2017). Despite these positive outcomes, there are still certain shortcoming associated with MICP technology which hinders successful industrial application (Seifan & Berenjian, 2019). It would be advantageous if the problems affecting MICP be addressed to widen its industrial potentials. Most MICP studies are still limited to laboratory tests which are relatively in small-scale (Wang et al., 2017). It is prudent that future studies move away from in-vivo experiments to in-situ trials for expansion of knowledge on how these uncontrollable environments would affect MICP process and microbial mechanisms.

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Undoubtedly, it will be a big challenge or impossible to transcend the optimized laboratory-conditions which favour biocalcification to the complex environmental factors. Inconsistencies between laboratory-scale and field-scale outcome could also possible arise due to various factors (i.e. different behaviour in porous media; different chemical composition; different kinetics; the presence of unfamiliar microbial communities and different utilized growth media) (Cunningham et al., 2019). However, it is necessary to have more field-scale projects for the enhancement of the existing data for MICP practicability and feasibility. More future in-situ tests will help address these rigorous scale-up treatment challenge affecting MICP technology. From an economic viewpoint, the current cost for MICP process should be significantly minimized. Almost all researchers utilize pure-grade reagents for MICP studies. Understandably, this is to promote repeatability and quality control. However, to ensure MICP is commercially implemented, alternative materials which are feasible and cost-effective should be strongly utilized. This could occur if materials such as commercially procurable technical-grade reagents (i.e. growth media and chemicals) are used to replace the expensive analytical-grade reagents. Preliminary findings (i.e. lactose mother liquor for bacterial cultivation and eggshell for CO3 precipitation) in the literature has shown that utilizing materials which are not high-purity showed comparable growth rate, urease activity and compressive strength performances to that of laboratory-grade reagents (Achal et al., 2009; Choi et al., 2016).

More so, it is prudent that future MICP studies work on large-scale microbial cultivation for in-situ studies. There are sufficient data on biomass production, urease activity and CaCO3 precipitation for ureolytic microorganisms under laboratory conditions. Future works must demonstrate how low-cost scalability process can be done. Data from the literature on small- scale bacterial cultivation could lead to an improvement in parameters necessary for scale-up parameters (Wang et al., 2017). It is understandable that right now there is insufficient information available to guide future researchers on how to cost-effectively scale-up the production of ureolytic bacterial cells. The use of cultivation medium, chemicals and reactors for field-scale applications do not have to be of the same grade as those used under laboratory- conditions (Zhu & Dittrich, 2016). Researchers should investigate the plausibility of using custom-built reactor for large-scale microbial production. This may tremendously suppress the high-cost of MICP process and promote the implementation of ureolytic bacterial cells to treat a large area.

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Several other issues are affecting MICP technology and strongly need to be resolved soon.

Scaling-up MICP treatment to mega level with heterogeneity of CaCO3 distribution along with soil matrix, determining the most efficient MICP treatment method to enhance biocalcification, limiting possible clogging formations which occurs during MICP treatment and developing suitable strategies to avoid or control ammonia production which occurs as a by-product during ureolysis (Mujah et al., 2017; Seifan & Berenjian, 2019; Wang et al., 2017).

2.12. Conclusions This study provides a state-of-the-art review of MICP technology for biotechnological and geotechnical engineering applications. Discussion on biomineralization, MICP pathways, urease sources and mechanisms, CaCO3 crystal polymorphs and soil biocementation were introduced in this study. In-depth and update of potential MICP applications were carefully reviewed. Also, challenges affecting MICP technology and future directions were provided. Based on this review, it can be concluded that MICP technology has tremendous potential which could help resolve numerous environmental problems. The continuous use of essential microorganisms with little or no ecological implications will enhance the biotechnological implication in an engineering discipline to perform sustainable products and treatment. If the earlier discussed limitations affecting MICP can be overcome, commercialization of MICP technology will be possible and prodigious. While researchers need to determine ways of resolving the problems inhibiting the industrial potential of MICP technology, the subsequent chapters (Chapter 3, 4 and 5) in this thesis investigates how the high-cost problem of MICP process can be minimized. This was done to enhance the feasibility of scaling-up the production of ureolytic bacteria for in-situ soil biocalcification.

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Chapter 3

Cultivation of Sporosarcina pasteurii strain using food- grade yeast extract medium

The contexts of this chapter presented herein contain peer-reviews and editorial edits. The paper was reprinted from Biocatalysis and Agricultural Biotechnology, Omoregie, A. I., Hei, N. L., Ong, D. E. L. & Nissom, P. M. (2019), ‘Low-cost cultivation of Sporosarcina pasteurii strain in food- grade yeast extract medium for microbially induced carbonate precipitation (MICP) application’, 17, 247-255. https://doi.org/10.1016/j.bcab.2018.11.030

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Biocatalysis and Agricultural Biotechnology

journal homepage: www.elsevier.com/locate/bab

Low-cost cultivation of Sporosarcina pasteurii strain in food-grade yeast extract medium for microbially induced carbonate precipitation (MICP) T application ⁎ Armstrong Ighodalo Omoregiea, , Lock Hei Ngua, Dominic Ek Leong Ongb, Peter Morin Nissoma a School of Chemical Engineering and Science, Swinburne University of Technology, Sarawak Campus, Jalan Simpang Tiga, 93350 Kuching, Sarawak, Malaysia b School of Engineering and Built Environment, Griﬃth University, 170 Kessels Road, Nathan, Queensland 4111, Australia

ARTICLE INFO ABSTRACT

Keywords: Sporosarcina pasteurii is a well-known ureolytic microbial species that proficiently induces the deposition of Bacterial cultivation calcium carbonate through microbially induced carbonate precipitation (MICP) process for various biotechno- Sporosarcina pasteurii logical and engineering purposes. In view to resolving the concern on high-cost bacterial cultivation due to the Urease activity conventional use of laboratory-grade growth medium for MICP studies, an inexpensive food-grade yeast medium Media optimization was investigated in this current study for its feasibility to serve as a suitable alternative media for bacterial Microbially induced carbonate precipitation growth, urease activity and calcium carbonate precipitation. The effect of different media concentration and (MICP) initial pH medium on biomass production and urease activity were determined. The performance of this low-cost media was also compared with eight laboratory-grade media (nutrient broth, yeast extract, tryptic soy broth, luria broth, fluid thioglycollate medium, cooked meat medium, lactose broth and marine broth). Results in this − current study showed cultivation in low-cost media at 15 g L 1 (w/v) and initial pH 8.5 of the food-grade yeast media both constituted the highest biomass concentration and urease activity when supplemented with urea (4%, w/v). Comparison of the food-grade media with laboratory-grade media indicated that bacterial cultivation cost was significantly reduced to 99.80%. After the biomineralization test, X-ray diffraction (XRD) analysis was

used to confirm the elemental composition of CaCO3 and polymorphs which were identified as calcite and vaterite. These findings suggest the food-grade yeast extract can serve as a potential candidate for bacterial cultivation in MICP application from the perspective of cost reduction.

1. Introduction process has been widely studied for the soil strengthening and stabilization, remediation of concrete cracks, restoration of limestone sur- Microbially induced carbonate precipitation (MICP) refers to the faces, mitigation of soil erosion, treatment of industrial wastewater and precipitation of calcium carbonate (CaCO3) which involves the com- remove heavy metals (Burbank et al., 2013; De Muynck et al., 2010; Fu bination of microbial and various biochemical activities (Bosak, 2011). and Wang, 2011; Işik et al., 2010; Sarda et al., 2009; Van Tittelboom MICP process via ureolysis (urea hydrolysis) utilizes microorganisms et al., 2010; Whiﬃn et al., 2007). MICP via ureolysis process can also be such as Sporosarcina pasteurii (S. pasteurii) to produce urease enzyme used for biosensor, agriculture and healthcare applications (Phang that facilitates the breakdown of urea (CO[NH2]2) for the generation of et al., 2018). Nonetheless, MICP is frequently employed to produce bio- + −2 ammonium (NH4 ) and carbonate (CO3 ) ions (Hammes and calcifying agent (CaCO3) which is used as a construction and building Verstraete, 2002). In the presence of calcium ions (Ca+2) such as cal- material to improve the geotechnical properties of soils. −2 +2 cium chloride (CaCl2), CO3 reacts with Ca to form CaCO3 (Stocks- Despite numerous attainable outcomes of this technology at la- Fischer et al., 1999). boratory-scale, MICP is still considered expensive for commercial im- In recent decades, microbial urease for MICP process has fervently plementation or ﬁeld-scale applications (Mujah et al., 2017). One of the seen an increased relevance as a catalyst for the precipitation of calcium reasons is due to the nutrient medium used in the biotechnological carbonate (CaCO3) for various applications in biotechnological and process for bacteria cultivation (Achal et al., 2009). The use of la- engineering disciplines. Investigations on use of ureolysis-driven MICP boratory-grade growth media (i.e. nutrient broth, yeast extract and

⁎ Corresponding author. E-mail address: [email protected] (A.I. Omoregie). https://doi.org/10.1016/j.bcab.2018.11.030 Received 17 October 2018; Received in revised form 29 November 2018; Accepted 29 November 2018 Available online 01 December 2018 1878-8181/ © 2018 Elsevier Ltd. All rights reserved. A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 247–255 tryptic soy broth) are a major cost factor for bacterial cultivation due to (Reyes et al., 2009). Similarly, laboratory-grade yeast extract (LG-YE) − their high cost that can reach up to 60% of the total production cost media (20 g L 1, BD BACTO™) and urea (4%, w/v) were prepared (Kristiansen, 2006). alongside the FG-YE media to serve as a comparative growth media for To date, only limited studies in the literature have reported the use test on bacterial growth profile, pH profile and urease activity. The of alternative nutritional sources to cultivate ureolytic bacteria for bacterial cultures were aerobically incubated at 30 °C for 60 h with MICP application. The use of waste materials such as chicken manure shaking (130 rpm) and monitored regularly (12 h interval) using dif- effluent, corn steep liquor, lactose mother liquor and activated sludge ferent parameters (optical density, pH and conductivity). The optical from agricultural, dairy and bakery industries have been the focus of density (OD) and pH were both measured hourly until the end of the alternative nutritional source (Achal et al., 2010, 2009; Cuzman et al., incubation in order to evaluate the bacterial development (biomass) 2015a; Joshi et al., 2018; Whiffin, 2004; Yoosathaporn et al., 2016). while the conductivity was only measured at the end of the incubation These attempts are mostly performed to limit the economic challenges to determine the urease activity. faced for cultivating ureolytic bacterial in large volume (Anbu et al., 2016). Irrespective of the promising findings which have been reported 2.3. Growth and pH profiles in the aforementioned studies for bacterial cultivation and also help minimize accumulative environmental wastes through recycling Bacterial growth was assessed by measuring the OD which served as (Yoosathaporn et al., 2016). Unfortunately, most recent contemporary a biomass concentration indicator for ureolytic bacteria (Harkes et al., studies on MICP still make use of conventional growth media to grow 2010). The OD was measured by using a spectrophotometer (Thermo various ureolytic bacterial species. Scientific™ GENESYS™ 20) at a wavelength of 600 nm. Before mea- There are some suggestive drawbacks that can hinder the use of surement of the OD, un-inoculated growth medium which served as waste materials for MICP applications which includes: (i) limited blanks were used to calibrate the spectrophotometer. Three millilitre quality control and processing (Williams et al., 2016); (ii) unavailability (3 mL) of the aliquot was sampled from the bacterial cultures and in localized regions; (iii) insufficient bulk quantity (i.e. large volume) placed into clean 10 mm cuvettes and the OD values were recorded. and (iv) transportation to site. Hence, there is a need to explore other Additionally, a pH meter (SevenEasy™–Mettler Toledo) was used to types of nutrition sources which are easily accessible at low or no cost measure the acidity or alkalinity of the bacterial culture in relation to suitable for MICP application. The aim of this present study was to the number of hydrogen ions (H+) or hydroxyl ions (OH-). The pH determine the feasibility of cultivating S. pasteurii strain in an eco- electrode was first calibrated with pH 4, 7 and 10 buffer solutions nomical food-grade yeast extract (FG-YE) medium and investigate its (Sigma-Aldrich) before immersing into the medium to obtain the re- effect on bacterial production, urease activity and biocalcification. The spective pH values of the bacterial cultures after incubation. performance of FG-YE was compared with eight laboratory-grade growth media. XRD analysis was further used to determine the ele- 2.4. Urease activity mental compositions and identify the polymorphs of CaCO3 precipitates − after biocalcification test. Urease activity (mM urea hydrolysed·min 1) of the bacteria cultures were determined immediately after sampling through electric con- 2. Materials and methods ductivity measurement (Whiffin, 2004). The urease reaction involves the hydrolysis of non-ionic substrate urea to ionic products that lead to 2.1. Ureolytic bacteria and cultivation an increasing proportionate conductivity under standard conditions (Al-Thawadi, 2008). For the urease assay, 10 mL of overnight grown Ureolytic bacterial S. pasteurii NB28(SUTS) (GenBank accession no. bacterial cultures were inoculated into sterile beakers (250 mL capa- KX212192.1) isolated by Omoregie et al. (2017) from limestone cave city) containing 90 mL of 1.5 M urea solution. The respective con- − sample was used in this study due to its high urease activity. The ductivity values (mS.cm 1) from the bacterial-urea solution were re- bacterium was revived on Petri dishes containing sterile tryptone soya corded for a duration of 5 min at 25 ± 2 °C (Whiffin et al., 2007). The − agar (40 g L 1, HiMedia, Laboratories Pvt. Ltd) and urea (4% w/v, probe of the conductivity meter (Milwaukee, MI806) was immersed Merck, Shd. Bhd). The Petri dishes were incubated (MMM incucell 55) into the beakers containing the solution to obtain the conductivity at 30 °C for 48 h under aerobic conditions and the grown colonies were readings. At the end of the assay, the conductivity variation rate − − subsequently sub-cultured until pure isolates were obtained. A loopful (mS·cm 1·min 1) was then determined from a slope of the plotted of the bacterial colony was then taken and inoculated into a universal graph using values of conductivity measurement against time − bottle containing sterile 10 mL tryptic soy broth (30 g L 1 Becton (Omoregie et al., 2017). In the measured range of activities, 1 mS − − Dickinson Shd. Bhd) and urea (4%, w/v). The broth cultures were in- min 1 corresponds with a hydrolysis activity of 11 mM urea min 1 ® cubated (CERTOMAT CT plus – Sartorius) at 30 °C for 24 h with (Harkes et al., 2010). shaking (130 rpm). The bacterial cultures were then temporarily pre- served in the fridge (4 °C) at the end of the incubation until when 2.5. Effect of media concentration needed. All the standard bacterial inoculum used for subsequent experiments were not stored for more than a month (Cuzman et al., Different concentrations of the FG-YE media were evaluated to es- 2015b). tablish the appropriate amount needed for maximum bacterial growth and urease production. The concentration of the media studied ranged − 2.2. Evaluation of FG-YE medium from 5 to 25 g L 1 (w/v). In order to understand the influence of urea addition in the inexpensive FG-YE, cultivation medium with afore- The ability of S. pasteurii NB28(SUTS) to grow in alternative low- mentioned concentration was prepared and grouped into two; (i) cost media was tested by cultivating it in FG-YE manufactured by Angel medium containing urea (4%, w/v) and (ii) medium without urea. Yeast Co., Ltd. (Supplementary materials, Fig. A1) which is habitually When the nutrient medium was sterilized, only then was urea added in used for baking and cooking purposes. The nutrition contents of the the selected culture flasks after the medium had cool down. The culti- media and physiochemical properties are both shown in Supplementary vation medium was therefore inoculated with overnight grown bac- materials, Table A1-A2. The ureolytic bacterium was grown in standard terial culture and incubated at 30 °C for 24 h with shaking (130 rpm). 250 mL Erlenmeyer culture flasks containing 125 mL of FG-YE The conductivity and OD were measured at the end of the cultivation − (20 g L 1) and supplemented with urea (4%, w/v). The initial pH of the period. The optimum media concentration that promoted the highest growth medium was adjusted to 8.0 using 0.1 M NaOH or 0.1 M HCl enzyme activity and OD was selected then used for subsequent

248 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 247–255 experiments unless where stated otherwise.

2.6. Eﬀect of initial pH medium Cost (US$) 3.56 7.83 5.42 4.09 0.27 134.06 2.19 b 1.59

The inﬂuence of initial pH medium for the FG-YE on the perfor- ) 1 mance of the ureolytic bacteria was studied ranging from 5.0 to 11.0 − with an interval of 0.5. The bacteria cultures were incubated at conditions previously mentioned (subsection 2.5. Eﬀect of media concentration). The OD, pH and urease activity of the bacterial cultures in Quantity (g·L 20 37.4 29.5 25 15 125 13 a various medium were measured at the end of the incubation period. 13

2.7. Comparison with various laboratory-grade growth medium

A variety of eight laboratory-grade cultivation media were selected LG-YE LB MB FTM TSB 30 3.68 LUB NB and used in this study for comparison with the inexpensive FG-YE on Media acronym the basis of bacterial growth and urease activity performances. The ), ), 1 1

standard media selected in this experiment which are typically used for − ). CMM − −1 1 a wide range of bacterial cultivation are: LG-YE (20 g L ,BD − ), sodium

− ) and

1 1

™ 1 BACTO ), nutrient broth (NB) (13 g L , Merck), luria broth (LUB) ), yeast − 1 − − − 1 1 ™ ﬂ −

(25 g L , Merck), tryptic soy broth (TSB) (30 g L , BD DIFCO ), uid ), sodium −1 1 thioglycollate medium (FTM) (29.5 g L , BD BBL™), cooked meat − − medium (CMM) (125.0 g L 1, BD DIFCO™), lactose broth (LB) − − (13 g L 1, Merck) and marine broth (MB) (37.4 g L 1, BD DIFCO™). − Alongside these high-cost media, the FG-YE medium (15 g L 1, Angel ). ). 1 1

Yeast Co., Ltd.) was also prepared and each of the growth media was − ), magnesium chloride (5.9 g L − 1 ), sodium bicarbonate (0.16 g L − 1 ), sodium chloride (5 g L ). supplemented with urea (4%, w/v). The bacteria were cultivated in ) and sodium chloride (2.5 g L 1 1 1 − ), sodium silicate (0.004 g L − − − ), sodium chloride (2.5 g L ﬂ 1

Erlenmeyer asks (125 mL working volume) incubated at 30 °C for 24 h 1 − with shaking condition (130 rpm). The optical density, pH and urease − -ribonucleotide) and sodium chloride. FG-YE ′ activity of the bacteria in all nine growth media was determined as described in the monitoring parameters sub sections. The characteristic ). of the ingredients and cost analysis for all the growth media used in this 1 ), monopotassium phosphate (2.75 g L ), dextrose (2 g L 1 ), glucose (2.5 g L − 1 1 − − study are presented in Table 1. ) and agar (0.75 g L − 1 ) and yeast extract (1.5 g L 1 − − ), boric acid (0.022 g L ), sodium chloride (19.45 g L 1 2.8. Biomineralization test 1 − − ), potassium chloride (0.55 g L 1 ), anhydrous dextrose (5 g L ) and disodium phosphate (0.008 g L − 1 1

ﬁ − −

A modi ed biomineralization method of Arias et al. (2017) was used ). 1 in this experiment to test the bio-calcifying eﬀect of the selected growth − ), resazurin (5 g L media with the use of the ureolytic bacteria. The overnight grown 1 ) and yeast extract (5 g L − 1 − bacterial cultures were inoculated in falcon tubes containing ce- ), beef extract (1.5 g L −1 1 mentation solution which consisted of urea (20 g L , Merck, Shd. Bhd) − ), ferric citrate (0.1 g L −1 1 − and calcium chloride (20 g L , HiMedia). To constitute bacterial ), peptic digest of soybean (3 g L ), L-cystine (0.5 g L ), dipotassium phosphate (2.75 g L 1 1 −1 1 − − ). −

growth during the MICP process, the cultivation media (5 g L ) men- 1 − ), strontium chloride (0.034 g L ), peptic digest of animal tissue (20 g L ), calcium chloride (1.8 g L 1 1 tioned in the aforementioned subsection (2.7. Comparison with various 1 − − − laboratory-grade growth medium). The cementation reagents were

prepared in deionized water before transferring to beakers (500 mL). ), ammonium nitrate (0.0016 g L 1 − ), sodium chloride (10 g L

The growth media were sterilized with the autoclave machine while the ), lactose (5 g L 1 1 ) and sodium lauryl sulfate (0.1 g L ), sodium chloride (5 g L ), yeast extract (1 g L 1 − 1 ), sodium thioglycolate (0.5 g L 1 − 1 −

urea and calcium chloride were ultraviolet light-sterilized. These che- − − micals were added into the growth medium after being cooled down in − order to avoid degradation of the chemicals. These cementation solutions (45 mL) were then transferred into sterile falcon tubes and in- uoride (0.0024 g L yeast extract, maltodextrin foodpeptides, addictive free (monosodium amino glutamate acids, and purine disodium base, 5 pyrimidine base and hydro-soluble vitamin B group. tryptone (10 g L peptone (5 g L peptone (5 g L tryptose (20 g L Composition chloride (5 g L pancreatic digest of casein (15 g L extract (5 g L magnesium sulfate (3.24 g L ﬂ potassium bromide (0.08 g L oculated with 5 mL of overnight grown bacterial cultures. The assay dipotassium phosphate (2.5 g L was performed in triplicate and the falcon tubes were incubated for 72 h at 32 °C with no shaking. At the end of the incubation period, the tubes were kept in a centrifuge machine (Eppendorf, 5804R) 10,000g

ﬄ ﬄ / for 5 min to separate the precipitates from the e uents. The e uents ™ were transferred into separate sterile falcon tubes (50 mL) and their / #226730) heart tissue granules (98 g L ™ respective pH values measured. The precipitates were washed with / #211825) pancreatic digest of casein (17 g L ™

distilled water, dried at 60 °C for 4 h and then weighed. / #212750) / #2216) ™ ™ /#70149) ® /#70142) ®

2.9. XRD analysis /#L3522) ®

For determination of elemental composition and crystalline phase of precipitated samples after biocalciﬁcation test, four sets of precipitated samples which were induced by ureolysis process and separate treat- #211260) All growth media used in this study were produced in Malaysia. Exchange rate MYR 4.15 = US$ 1.0. ment cementation solutions containing FG-YE, LG-YE, TSB and NB were Quantity of all the media added except for FG-YE were based on manufacturer's instruction. a b Nutrient broth (Merck Growth media Cooked Meat Medium (BD DIFCO Fluid Thioglycollate Medium (BD BBL Tryptic Soy Broth (BD DIFCO Yeast extract (Angel Yeast/FB00) Yeast extract (BD BACTO Lactose Broth (Merck Marine Broth (BD DIFCO Luria broth (Merck

respectively selected for XRD analysis. The dried precipitated samples Table 1 Growth media and cost analysis.

249 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 247–255

− were first transferred into newly sterilized falcon test tubes (50 mL) FG-YE a constant growth rate of 0.05 h 1 and a doubling time of before they were sent to Quasi-S Sdn. Bhd. Malaysia for XRD analytical 14.23 h at the end of the growth study. Noteworthily, the maximum OD service. The XRD spectra were obtained using X-ray Diffractometer value (1.21) for LG-YE occurred at 42 h incubation period, while that of X′Pert3 Powder XRD equipment (Panalytical) scanning the fine-pow- FG-YE occurred at 54 h incubation period with OD of 1.04. The pH dered samples from 5° to 80° 2θ with Cu anode having 40 kV and 35 mA profile of the ureolytic bacteria in both media showed a simultaneous running parameters. The analysed components of the precipitated increase towards alkaline level as the growth profile increased which is samples were identified by comparing them with standards established an indication of ureolysis process. The final pH values of the bacterial by the International Centre for Diffraction Data (Al-Salloum et al., culture in LG-YE and FG-YE medium were 9.16 and 9.17, respectively. 2017; Navdeep Kaur Dhami et al., 2017a). There were noticeable slight differences between the growth profile of the ureolytic bacteria when grown LG-YE and FG-YE, it is possible this 2.10. Statistical analysis could be influenced by the yeast extract components such as maltodextrin food additive and salt contents present in the FG-YE medium. All the experiments in this work were carried out in triplicates. The Yeast extract typically contains abundant of amino acids, peptides, experimental results were analysed using the statistical analysis soft- carbohydrates, salts, vitamins and minerals which are necessary for ® ware GraphPad Prism (version 7) to determine the significance of microbial cell cultivation (Li et al., 2011). Commercially manufactured differences between the means groups obtained in this study. One-way yeast extract for bakery and cooking purposes often contains other in- analysis of variance (ANOVA) and Tukey-Kramer post hoc test were gredients such as flavouring agent in soup, snacks and canned foods for performed using Minitab® (version 18). The significance level was set at food industry containing (Milić et al., 2007). Nevertheless, the growth P < 0.05. and pH profile showed that the ureolytic bacteria could adapt in the low-cost media. 3. Results and discussion 3.2. Effect of media concentration 3.1. Evaluating FG-YE medium For mass production of ureolytic bacteria, it is important to use The growth and pH profiles of S. pasteurii NB28(SUTS) which was inexpensive media which can support bacterial growth and high urease studied up to 60 h at 30 °C in both FG-YE and LG-YE medium yielded activity (Cuzman et al., 2015b). In order to optimize biomass and ur- similar trends in relation to their respective OD (Fig. 1). LG-YE had a ease production in the inexpensive FG-YE to favour cost reduction, −1 ff constant growth rate of 0.04 h and a doubling time of 16.96, while several cultures of the ureolytic bacteria were grown with di erent − concentrations (5–25 g L 1). It is important to note that some low-cost LG-YE FG-YE media are often subjected to lower quality control and reproductivity, hence its effect on urease production should be considered (Cuzman A 1.5 et al., 2015b). The results in Fig. 2A and Fig. 3 showed that the re- ) spective maximum OD (1.02) and urease activity (20.16 mM urea hy- − 600 drolysed.min 1) for the ureolytic bacteria in the FG-YE medium con- − taining urea was produced in 15 g L 1 media concentration, while −1 1.0 5gL produced the lowest OD (0.87) and urease activity (10.43 mM − urea hydrolysed.min 1). On the other hand, the maximum and lowest OD (1.56 and 1.15) for the bacteria in the FG-YE medium without urea − − (Fig. 2B) were respectively produced in 10 g L 1 and 5 g L 1 media 0.5 concentrations, while the maximum and lowest urease activity (8.72 − and 6.92 mM urea hydrolysed min 1) were respectively produced in − − 20 g L 1 and 5 g L 1 as shown in Fig. 3. ANOVA and Tukey-Kramer fi ff ff Optical density (OD post hoc test showed there were signi cant di erences on the e ect of 0.0 media concentration among the group means when the bacteria were 0 1224364860 grown in FG-YE medium containing urea (P-value = 3.49E-4) and Time (h) without urea (P-value = 2.13E-9). Additionally, the statistical analysis for urease activity also showed significant differences among the group means when cultured in FG-YE medium with urea (P-value is 1.26E-5) B 9.5 and no urea (P-value = 3.41E-6).

3.3. Eﬀect of initial pH medium

9.0 The growth, pH and urease activity of the ureolytic bacteria studied in an FG-YE medium having different initial pH medium is shown in Fig. 4. pH 8.5 had the highest OD (1.43) and urease activity (22.84 mM − urea hydrolysed.min 1), but when the initial pH medium was at 11, it pH value 8.5 had the highest pH value (9.41) which occurred at the end of the incubation period. On the other hand, the lowest pH values (9.14) and − urease activity (14.78 mM urea hydrolysed min 1) were observed when 8.0 the initial pH medium was at pH 5.0 with the lowest OD of 1.15. The 0 1224364860 initial pH of the culture media has a huge effect on the growth of microbes, for most bacteria there is a correlation as the growth rate in- Time (h) creases, the pH approaches its optimum value (Basu et al., 2015). These results showed that S. pasteurii NB28(SUTS) strain could grow and Fig. 1. The growth profile (A) and pH profile (B) of S. pasteurii NB28(SUTS) produce urease when FG-YE was adjusted to acidic or alkaline condi- −1 −1- cultivated in LG-YE (20 g L ) and FG-YE (20 g L ). tions but for maximum biomass and urease activity, the initial pH

250 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 247–255

A 1.2 Urea No urea 25 ) a ab

b ab ) 600 c -1 a 0.8 20

15 ab ab 0.4 b 10 c a a Optical density (OD

Urease activity a b 0.0 5 c -1 -1 -1 -1 -1

.L L (mM urea hydrolysed.min 5g.L 10 g.L 15 g 20 g. 25 g.L 0 -1 -1 -1 -1 -1 Media concentration (w/v) 5g.L 10 g.L 15 g.L 20 g.L 25 g.L Media concentration (w/v) B Fig. 3. Inﬂuence of the low-cost media concentration with and without urea on 2.0 urease activity. The alphabets indicate statistically signiﬁcances (P < 0.05) as ) determined by one-way analysis of variance (ANOVA) with means comparison 600 a using Tukey-Kramer post hoc test. 1.5 b c d e Fig. 5 showed that among all the growth media used to cultivate the bacteria, TSB has the highest OD (1.07) for biomass production while 1.0 MB had the lowest OD (0.81). In addition, CMM was shown to have the highest pH value (9.25), while MB had the lowest pH value (9.08). The 0.5 result also showed that TSB had the highest urease activity (18.71 mM − urea hydrolysed.min 1), while LB had the lowest urease activity −1

Optical density (OD (10.85 mM urea hydrolysed.min ). FG-YE had comparable biomass 0.0 (0.99), pH (9.20) and urease activity (14.77 mM urea hydro- -1 -1 -1 -1 -1 − lysed.min 1) with these eight selected standard media under the same 5g.L 10 g.L 15 g.L 20 g.L 25 g.L growth conditions. The statistical analyses showed there were significant differences for biomass production (P-value is = 1.118E-8), pH Media concentration (w/v) values (P-value is = 8.806E-6) and urease activity (P-value is = 2.175E-12) when the group means were compared between each other. Fig. 2. Influence of the low-cost media concentration with urea (A) and without In terms of cost as shown in Table 1, FG-YE media was the cheapest urea (B) on biomass production. Vertical error bars indicate the means ± SE of (0.29 US$ per litre) media, while CMM was the most expensive (143.43 three independent repetitions. The alphabets indicate statistically significances (P < 0.05) as determined by one-way analysis of variance (ANOVA) with US$ per litre) media. By using the FG-YE media for the cultivation of S. means comparison using Tukey-Kramer post hoc test. pasteurii as a replacement to conventional microbiological media, the bacterial production cost which was significantly reduced ranged between 82.80% and 99.80%. Although the cost of laboratory-grade medium has to be set at 8.5. This finding is supported by other studies growth media is excessively expensive when considered for large-scale which have indicated that pH 8–9 are the optimum conditions to pro- cultivation of ureolytic bacteria (Cheng and Cord-Ruwisch, 2013). The duce good levels of urease activity (Bang et al., 2001; Stocks-Fischer low-cost FG-YE media which is commercially available and sold for US et al., 1999). It was also noticed that at the end of each incubation $18.00 per kilogram makes it remarkably affordable considering that period, despite adjusting the initial pH medium of the broths between 5 the laboratory-grade growth media used in this current study cost be- and 11, the bacteria were able to regulate the micro-environment to its tween US$122.00–1073.00 per kilogram. Clearly, the use of MB media near preferred optimum pH value. It is possible that the ureolytic to cultivate ureolytic bacteria for MICP applications is impracticable bacteria was able to secrete different compounds which allowed it to due to its extreme high cost, but the use of other laboratory-grade alter the growth medium's pH, hence to a much-favoured pH state. media is often common, particularly TSB, NB and AG-YE media. For Bacteria are able to manipulate their environment to maintain a certain mass production of ureolytic bacteria, it is important to use inexpensive cytoplasmic pH which correlates with their optimal functional and media which can support bacterial growth and high urease activity structural integrity (Padan et al., 2005). The statistical analysis for (Cuzman et al., 2015b). In order to perform biocalcification treatment biomass production (P-value is = 0.00), pH value (P-value is = 0.00) of building materials, the price of growth medium and nutrients to and urease activity (P-value is = 1.51E-16) on the effect of different cultivate ureolytic bacteria for the concentration of 2–3 g per meter initial pH medium showed that there were significant differences be- square have been estimated to cost up to US$240.00 per kilogram (De tween the group means. Muynck et al., 2010). Hence, the use of low-cost growth media at a − concentration of 15 g L 1, high level of urease activity and bacterial 3.4. Comparison with laboratory-grade media production are feasible with the prospect of replacing laboratory-grade growth media. To further determine the suitability of using the FG-YE media as an alternative nutrient source, eight standard growth media were selected and tested for comparison with the low-cost FG-YE media. The result in

251 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 247–255

A 1.6 A 1.4 ) a ) 600 1.2 1.4 600 a b bc abc abc abc ab e de de cd cde c bc f 1.0 d f f f d 1.2 g 0.8

0.6 1.0 0.4 Optical density (OD 0.2

0.8 Optical density (OD 5 6 7 8 9 .5 .5 5.5 6.5 7.5 8.5 9 10 11 0.0 10 InitialpHofmedium E MB NB LB BB CMM TSB FTM L LG-YEFG-Y B 10.0 Various growth medium B 9.5 10.0 pH value 9.0 9.5

8.5 pH value 9.0 5 6 7 8 .5 9 5.5 6.5 7.5 8 9.5 10 0.5 11 1 Initial pH of medium 8.5 C 25 M ) a MB NB SB LB -1 CM T FTM LBB b b b bc LG-YEFG-YE 20 de cd cd de e Various growth medium f f f 15 C ) 20 a 10 -1 a

Urease activity b bc 5 bc 15 f e de (mM urea hydrolysed.min 0 cd

5 6 .5 7 8 9 5 5.5 6 7.5 8.5 9.5 10 11 10 10. Initial pH of medium

Urease activity 5 Fig. 4. Influence of various initial pH medium on the biomass production (A); pH (B) and urease activity (C) for S. pasteurii NB28(SUTS). Vertical error bars indicate the means ± SE of three independent repetitions. The alphabets in- (mM urea hydrolysed.min 0 dicate statistically significances (P < 0.05) as determined by one-way analysis of variance (ANOVA) with means comparison using Tukey-Kramer post hoc -YE MB NB LB CMM TSB FTM LBB test. LG FG-YE Various growth medium 3.5. Biomineralization test Fig. 5. Comparison of different selected laboratory-grade growth media with the food-grade yeast extract media for their respective performances on growth Carbonate crystals were precipitated by the ureolytic bacteria in all (A); pH (B) and urease activity (C). Vertical error bars indicate the means ± SE the cementation solutions which contained different growth media as of three independent repetitions. The alphabets indicate statistically sig- part of essential cementation reagents required to induced calcification nificances (P < 0.05) as determined by one-way analysis of variance (ANOVA) after 72 h at 32 °C. As shown in Fig. 6, despite the similarity of the with means comparison using Tukey-Kramer post hoc test. weight contents of CaCO3 constituted by the different cementation so- −1 −1 lution, MB (6.76 g L ) produced the highest, while LB (2.80 g L ) reading. The FG-YE had comparable results with the rest of the samples produced the lowest precipitate. The effluents were collected and −1 having 5.79 g L of the estimated CaCO3 precipitates and effluent with measured for pH readings showed that TSB (8.06) had the highest pH pH 7.61. The statistical analysis showed significant differences (P-value fi value after the biocalci cation process, while NB (6.84) showed to have is = 1.536E-12) among the group means. fi the lowest pH value. In addition, prior to the biocalci cation test, the MICP via urea hydrolysis is a straightforward biochemical process measured pH values for the cementation solutions showed that TSB which is regulated by the concentration of calcium ion, the con- (5.30) also had the lowest reading, while CMM (7.09) had the highest centration of dissolved inorganic carbon, the pH of the

252 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 247–255

A of ureolytic bacteria's microenvironment are often increased due to the 8 release of ammonium ions during urea hydrolysis. ) -1 a 3.6. XRD analysis b bc bc 6 bc cd d d The four-powder crystal precipitated samples which were selected and tested for XRD analysis showed that the most intensive peaks were clearly observed at the angle of 29.8° 2θ as shown in Fig. 7. Calcite was

4 the only CaCO3 polymorph detected in each of the precipitated samples tested except for FG-YE which indicated the precipitates had calcite c (94.9%) and vaterite (5.1%) polymorphs of CaCO3. The XRD analysis precipitate (g.mL

3 also indicated that the intensity of the peaks for the minerals diﬀered 2 for each sample at the angle of 29.8° 2θ. TSB sample had the highest XRD spectrum with an intensity reaching up to 3400, while NB sample CaCO showed the lowest spectrum having an intensity of 1200. It's been

0 suggested that different morphological CaCO3 crystals formed are often M influenced by urease activity by strain-specific microorganisms and NB -YE -YE LB MB TSB CM FTM LBB LG FG alternatively the extracellular polymeric substance (EPS) protein produced by different microbial strains (Hammes et al., 2003; Park et al., Cementation reagents 2010). This is because EPS is known to specifically bind with Ca2+ and induce the type of CaCO3 polymorphs crystals which will the precipitated (Dhami et al., 2013a, 2013b). Before bacterial inoculation The XRD analysis for crystal minerals precipitated by ureolytic After bacterial inoculation bacteria in cementation solutions separately containing TSB, NB and B 10 AG-YE were selected alongside low-cost media because they are often used as part of cementation reagents for MICP studies (Al-Salloum a b b et al., 2017). Initially, the current authors hypothesized that CaCO 8 a c 3 a f e g polymorphism might be affected when the FG-YE is utilized as a ce- d h b mentation reagent. Thus, it was interesting that our result showed that d c c 6 the low-cost media induced both calcite (94.9%) and vaterite (5.1%) e e e polymorphs, while all the standard growth media all had only calcite. The result also showed that irrespective of the growth media used, 4 calcification will still occur and the formation of stabilized calcite might fi ff

pH of effluent not signi cantly be a ected if food-grade media is used. Previous stu- fl 2 dies have demonstrated that CaCO3 polymorphs can be in uenced by the composition of growth medium, substrate, pH and type of microbes (Dhami et al., 2013a, 2013b; Rodriguez-Navarro et al., 2012). This is 0 because the ionic strength of different growth media affects ureolytic bacteria in manners that supports or distress formation of mono- YE BB NB LB MB TSB CMM FTM G-YE L FG- L hydrocalcite (Bansal et al., 2016). The results in this present study thus strongly indicate that the FG-FE is capable of cultivating ureolytic

Cementation reagents bacteria and can be used to induce CaCO3 precipitates for biocement applications. ffl Fig. 6. The measurement of calcium carbonate contents (A) and pH of e uents It is desirable to precipitate calcite mineral as it is the most ther- (B). The biocalcification test was performed with cementation solutions con- modynamically stable polymorph of CaCO3 when compared to other taining cementation solutions supplemented with different growth media and less stable and less abundant polymorphic forms of CaCO (aragonite were incubated at 32 °C for 24 h incubation without shaking. Vertical error bars 3 and vaterite) (Boulos et al., 2014). The formation of vaterite is very rare indicate the means ± SE of three independent repetitions. The alphabets indicate statistically significances (P < 0.05) as determined by one-way analysis but elevated temperature or water rich in NaCl can accelerate its of variance (ANOVA) with means comparison using Tukey-Kramer post hoc transformation (Skinner and Jahren, 2007). Our current research study test. intended to achieve high calcite formation using FG-YE as part of cementation reagents used for MICP treatment. XRD analysis showed that low percentage of vaterite were performed alongside calcite when FG- microenvironment and availability of nucleation sites (Hammes and YE is used as part of cementation reagents for biomineralization test. It Verstraete, 2002). These factors play essential roles which favour −2 is suggested that this trace amount of vaterite could have been stimu- CO3 during the ureolysis-driven process. High bacterial cells con- lated by the salt contents present in the FG-YE media. Carbonate centration (0.8–1.2 OD) helps to enhance calcite deposits during the polymorphs have a high significance on the proficiency of bioce- MICP process because the higher cells increase urease concentration for mentation due to their varying characteristics of the polymorphs such urea hydrolysis (Langdon et al., 2000). In this study, the CaCO3 induced as stability and durability (Dhami et al., 2017a). Calcite enhances the by the ureolytic bacteria in the cementation solution containing FG-YE mechanical strength properties of soils via MICP process better than was similar to other standard media which indicates that the low-cost aragonite and vaterite (Dhami et al., 2016). Hence, it is often preferred media can serve as alternative reagent growth media in the cementation to have calcite contents for soil stabilization. However, the presence of solution. The results showed that none of the growth media affected low vaterite amount with high calcite contents after biocementation carbonate precipitation because they all allowed the precipitation of might not have a significant effect on strength improvement of soils. A CaCO3. The measured effluents indicated the presence of urease activity recent study by Dhami et al. (2017b) have shown vaterite precipitation after the biocalcification test because there was an increase in the pH of alongside calcite after MICP treatment on loose soils and the presence all cementation solution from acidic to an alkaline level. The pH levels of vaterite formations did not impede soil solidification. Thus, the use of

253 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 247–255

Fig. 7. X-Ray Diﬀraction analysis of four selected precipitated samples obtained after ureolysis process. Sample 1 (TSB); Sample 2 (AG-YE); Sample 3 (FG-YE) and Sample 4 (NB).

FG-YE media for bacterial cultivation and CaCO3 precipitation might Appendix A. Supporting information not be disadvantageous at upscaling stage of MICP application. Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bcab.2018.11.030. 4. Conclusions References In this present study, the feasibility of cultivating S. pasteurii NB28(SUTS) strain in food-grade yeast extract medium was in- Achal, V., Mukherjee, A., Basu, P.C., Reddy, M.S., 2009. Lactose mother liquor as an vestigated. The results obtained from this report showed the food-grade alternative nutrient source for microbial concrete production by Sporosarcina pasteurii. J. Ind. Microbiol. Biotechnol. 36, 433–438. https://doi.org/10.1007/s10295- yeast media was a good alternative source of nutrients for low-cost 008-0514-7. bacterial cultivation in a growth medium which can support the bio- Achal, V., Mukherjee, A., Reddy, M.S., 2010. Biocalcification by Sporosarcina pasteurii using corn steep liquor as the nutrient source. Ind. Biotechnol. 6, 170–174. https:// mass, urease activity and biomineralization of CaCO3. For the promo- doi.org/10.1089/ind.2010.6.170. tion of maximum biomass and urease activity in the inexpensive media, Al-Salloum, Y., Hadi, S., Abbas, H., Almusallam, T., Moslem, M.A., 2017. Bio-induction the effect of different media concentration and initial pH medium were and bioremediation of cementitious composites using microbial mineral precipitation − examined. The concentration of 15 g L 1 and initial pH 8.5 produced – a review. Constr. Build. Mater. https://doi.org/10.1016/j.conbuildmat.2017.07. the highest biomass and urease activity when the bacterial was culti- 203. Al-Thawadi, S.M., 2008. High strength in-situ biocementation of soil by calcite pre- vated in the media supplemented with urea (4%, w/v). The urease cipitating locally isolated ureolytic bacteria. Sch. Biol. Sci. 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Selective enrichment and production of highly urease active bacteria by non-sterile (open) chemostat culture. J. Ind. Microbiol. Biotechnol. This study was funded by Bachy Soletanche (Rueil-Malmaison, 40, 1095–1104. https://doi.org/10.1007/s10295-013-1310-6. ffi Cuzman, O.A., Rescic, S., Richter, K., Wittig, L., Tiano, P., 2015a. Sporosarcina pasteurii France) and School of Research O ce under Swinburne Sarawak use in extreme alkaline conditions for recycling solid industrial wastes. J. Biotechnol. Research Grants (SSRG 2-5162 and SSRG 2-5535). We also appreciate 214, 49–56. https://doi.org/10.1016/j.jbiotec.2015.09.011. Nurnajwani Senian (Ph.D. Candidate - Civil Engineering) for her as- Cuzman, O.A., Richter, K., Wittig, L., Tiano, P., 2015b. Alternative nutrient sources for sistance with procurement of the food-grade yeast extract media. biotechnological use of Sporosarcina pasteurii. World J. Microbiol. Biotechnol. 31, 897–906. https://doi.org/10.1007/s11274-015-1844-z.

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255 Supplementary materials

162

Figure A1: Food-grade yeast extract media.

163

Table A1: Nutrition contents of industrial-grade yeast extract Components Quantity (g.L-1)

Protein 29.4 Carbohydrate 51.4 Fat 0.0 Sodium 4.2

Table A2: Physico-chemical characteristics of industrial-grade yeast extract Composition Result

Total nitrogen, % 5.70

Amino nitrogen, % 2.80

Moisture, % 3.90

Sodium chloride, % 2.83

pH value (2% solution) 5.72

Texture Smooth

Solubility in water (2% solution) Complete

Appearance of powder Canary yellow

Appearance of non-sterilized solution Canary yellow

Appearance of sterilized solution Caramel brown

164

Chapter 4

Sand biocementation using Sporosarcina pasteurii strain and technical-grade cementation reagents

The contexts of this chapter presented herein contain peer-reviews and editorial edits. The paper was reprinted from Construction and Building Materials, Omoregie, A. I., Palombo, E., Ong, D. E. L., & Nissom, P. M. (2019), ‘Biocementation of sand by Sporosarcina pasteurii strain and technical-grade cementation reagents through surface percolation treatment method’, 228C, 116828. https://doi.org/10.1016/j.conbuildmat.2019.116828

165

Construction and Building Materials 228 (2019) 116828

Contents lists available at ScienceDirect

Construction and Building Materials

journal homepage: www.elsevier.com/locate/conbuildmat

Biocementation of sand by Sporosarcina pasteurii strain and technical-grade cementation reagents through surface percolation treatment method ⇑ Armstrong I. Omoregie a, , Enzo A. Palombo b, Dominic E.L. Ong c, Peter M. Nissom a,d a School of Chemical Engineering and Science, Swinburne University of Technology, Sarawak Campus, Jalan Simpang Tiga 93350 Kuching, Sarawak, Malaysia b Department of Chemistry and Biotechnology, Swinburne University of Technology, Hawthorn, VIC 3122, Australia c School of Engineering and Built Environment, Grifﬁth University, Brisbane, Queensland 4122, Australia d Sarawak Research and Development Council, C/O Ministry of Education, Science and Technological Research, LCDA Tower, off Jalan Bako, 93050 Kuching, Sarawak, Malaysia highlights

Low-cost growth medium was used to cultivate Sporosarcina pasteurii prepared in deionized water and tap water. Soil biocementation treatment was performed through surface percolation method. Analytical-grade cementation reagents can be replaced with technical-grade cementation reagents. Utilizing technical-grade reagents for sand biocementation resulted in 47 to 51-fold cost reduction. article info abstract

Article history: The use of microbially induced carbonate precipitation (MICP) to produce biocementitious material for Received 12 May 2019 soil stabilization has emerged in recent decades as a sustainable alternative approach to conventional Received in revised form 13 August 2019 methods. However, the use of standard analytical-grade reagents for various MICP studies makes this Accepted 28 August 2019 technology very expensive and unsuitable for field-scale consideration. In this present study, the feasibil- Available online 4 September 2019 ity of using commercially available and inexpensive technical-grade reagents for the cultivation of ureolytic bacteria and enhancement of soil stabilization was investigated. Low-cost growth media Keywords: prepared in deionized water and tap water were used to cultivate Sporosarcina pasteurii as a replacement Sporosarcina pasteurii to standard laboratory-grade media. Biocement treatment was carried out on sand columns using differ- Biocementation Crystal morphology ent concentrations (0.25–1.0 M) of technical-grade and analytical-grade cementation solutions via sur- Surface strength face percolation method. After 92 h of treatment, the columns were cured for 3 weeks at room Calcium carbonate temperature (26 ± 2 °C) before analysing their respective surface strengths, CaCO3 content, pH of efflu- Urease enzyme ents and sand microscopic structures. The results indicated that the growth of bacteria in low-cost cul- Nutrient tivation medium was similar to that observed in the standard cultivation medium. Surface strengths and Low-cost reagents CaCO3 contents of the consolidated samples were in the ranges of 11448.00 ± 69.00–4826.00 ± 00 kPa and 5.56 ± 1.15–33.24 ± 0.59%, respectively. Overall, the obtained results of the current study encourage future MICP studies to utilize commercially available technical-grade reagents for economical MICP field-scale trials. Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction for 6–8% of global anthropogenic CO2 emissions [1,2]. Greater awareness on the environmental and human impacts of conven- Cement is one of the most important binding agents used glob- tional cement production has inspired the need to investigate ally for construction and infrastructure purposes. Its production alternatives binders. Biocement (insoluble crystal) has recently from calcining limestone generates about 0.814 tons of carbon emerged as a new binding material which is produced through dioxide (CO2) per one ton of limestone and singularly accounts microbially induced carbonate precipitation (MICP) for soil stabilization [3]. It has the ability to bind porous materials together and enhance their mechanical properties (i.e. strength and ⇑ Corresponding author. E-mail address: [email protected] (A.I. Omoregie). impermeability). https://doi.org/10.1016/j.conbuildmat.2019.116828 0950-0618/Ó 2019 Elsevier Ltd. All rights reserved. 2 A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828

Soil improvement by biocement treatment methods often of using commercially available and inexpensive technical-grade involves the addition or injection of ureolytic bacteria such as Spor- reagents as replacements to the costly analytical-grade reagents osarcina pasteurii and cementation reagents (i.e. urea, calcium for bacterial cultivation and soil biocalcification. Through surface chloride and growth nutrients) into the soil [4]. Many microorgan- percolation treatment, different concentrations (0.25–1.0 M) of isms can precipitate calcium carbonate (CaCO3) but are not suit- cementation reagents prepared in tap water and deionized water able for MICP due to biosafety concerns related to their were used to calcify soil samples. The performance of technical- pathogenic capacity. Ureolytic bacteria such as Staphylococcus aur- grade reagents was compared with that of analytical-grade eus, Helicobacter pylori and Pseudomonas sp. are known to cause reagents. gastritis, ulcers, urinary tract infections, respiratory tract infections and nosocomial infections [5]. However, S. pasteurii strains are 2. Materials and methods often used in MICP studies because they are unlikely to cause human diseases. This bacterium is also often selected for bioce- 2.1. Ureolytic bacteria and cultivation medium ment investigations due to its highly active urease production and crystallization of CaCO3 [6]. The ureolytic bacterial strain S. pasteurii NB28(SUTS) (GenBank Biocement materials are able to bridge soil particles together accession no. KX212192.1) previously isolated by Omoregie et al. when the biocement materials are properly precipitated in the soil [32] from a local limestone cave sample was used throughout this matrix, eventually enhancing the mechanical properties [7,8]. current study due to its high urease activity, biocalcifying ability Unlike most established soil stabilization methods, biocementation and non-pathogenicity. The bacterium was revived on Petri plates can occur at ambient conditions with minimal emission of CO2 [9]. containing tryptone soya agar (40 g.LÀ1, HiMedia, Laboratories Pvt. Conventional soil stabilization practices which use mechanical (i.e. Ltd.) and urea (4% w/v, Merck, Shd. Bhd.). The bacteria were then soil compaction) or chemical (i.e. liquid silica) methods require incubated (MMM incucell 55) at 30 °C for 48 h under aerobic con- considerable labour, involve high viscosity materials with low soil ditions and sub-cultured until a pure culture was obtained. The penetrability and are hazardous to the environment and humans ureolytic bacterial cultures were stored at 4 °C and retrieved for [10,11]. On the other hand, biocementation is durable, less haz- use when needed [33]. All bacterial cultures used for the subse- ardous and suitable for deep penetration treatment with fine pores quent experiments were stored not more than a month [26]. Two [5]. Over the past two decades, biocementation via urea hydrolysis different cultivation medium designated Growth Medium-1 (GM- has been widely studied for numerous MICP applications which 1) and Growth Medium-2 (GM-2) were used throughout this cur- includes concrete and crack repair, soil consolidation and stabiliza- rent study to grow the ureolytic bacteria. GM-1 contained com- tion, and erosion control [12–17]. mercially available, inexpensive ingredients and was selected as Despite numerous laboratory-scale reports on soil biocalcifica- an alternative nutrient source to cultivate S. pasteurii, while GM- tion, the practical implementation of such methods is currently 2 contained standard and costlier ingredients typically used for limited [6]. It is crucial that the next phase of soil biocalcification laboratory experimentations to cultivate a wide range of microor- development should focus on the efficacy of scaling to field-scale ganisms. The compositions of both growth media and their respec- treatments [8]. Notwithstanding the promising results of the MICP tive cost analysis are presented in Tables 1 and 2, respectively. process from the literature, it is still considered expensive for prac- Both media were prepared in Schott bottles (1 L) containing deion- tical or commercial purposes. The feasibility of biocementation ized water and tap water. Physicochemical properties of the tap does not only rely on technical aspects for successful implementa- water (Table 3) were analysed by EnviC Laboratory Sdn. BhD tion, but it is also accompanied by economic challenges [7]. For Malaysia using the American Public Health Association standard example, the nutrient media (i.e. yeast extract) and synthetic method for examination of water and waste water samples. Sterile chemicals (urea and calcium chloride) used for bacterial cultiva- urea (by 0.45 lm filter sterilization) was added aseptically to the tion and treatment process are usually of high-grade. The use of Schott bottles after autoclaving to prevent chemical decomposition these high-purity reagents is essential for fundamental under autoclave condition [32]. The initial pH was adjusted to 7.00 laboratory-scale investigations, but their high cost makes their uti- using 0.1 M sodium hydroxide or 0.1 M hydrochloride acid after lization extremely costly for industrial applications [18]. In fact, sterilization [34]. 12.5 mL of overnight cultures were inoculated the use of conventional nutrient media for bacterial cultivation into separate 250 mL culture flasks containing 112.5 mL growth can contribute up to 60% of the total operating costs [19]. Addition- medium and incubated at 32 °C for 72 h with shaking (130 rpm). ally, the amount for these cementation reagents can reach up to US $9/m3, while the total cost for soil treatment ranges between US $25/m3 and US$500/m3 [16,20,21]. 2.2. Optical density and pH To date, there are limited studies in the literature that have explored the use of alternative cementation reagents for MICP. The performance of S. pasteurii NB28(SUTS) in both GM-1 and Seawater, eggshells, limestone from aggregate quarries, calcareous GM-2 was monitored (OD and pH profiles) during cultivation. sand, feed and calk powders have been investigated for their suit- The changes in cell density (biomass concentration) were moni- ability as calcium sources [1,22–25]. Cuzman et al. [26] and Fahmi tored by absorbance (optical density) using a spectrophotometer et al. [27] recently reported the use of fertilizer urea as a replace- (Thermo ScientificTM GENESYSTM 20) at a wavelength of 600 nm ment for synthetic analytical-grade urea, while Kuar et al. [28] (OD600). Three millilitres of the culture was sampled and placed and Chen et al. [29] showed that CO2 and pig urine could serve into clean 10 mm cuvettes before reading the values [35]. The as suitable substitutes for synthetic analytical-grade urea. Waste spectrophotometer was calibrated using un-inoculated growth materials such as corn steep liquor have also been studied as a pos- media (GM-1 and GM-2) as blanks before the optical density of sible replacement for the standard growth medium used to culti- bacterial cultures grown in these two respective medium was mea- vate ureolytic bacteria [12,27,30]. Unfortunately, most sured. A pH meter (SevenEasyTM–Mettler Toledo) was used to mea- contemporary biocalcification studies still make use of costly sure the pH of the bacterial cultures. Prior to measuring the pH analytical-grade chemicals and microbiological media. It is crucial values, the pH electrode was calibrated with pH 4.01, 7.00 and that future investigations explore other cementation reagents 10.30 buffer solutions (Sigma-Aldrich) [35]. The optical density which are either of low or no cost and can be easily accessible and pH values were both monitored regularly (6 h interval) until [31]. The aim of this current study was to evaluate the feasibility the end of the incubation period (48 h). A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 3

Table 1 The two nutrient media used to cultivate S. pasteurii NB28 (SUTS).

Culture Ingredients (concentration, supplier) Initial medium pH aGM-1 Yeast extract (15 g.LÀ1, Urea (40 g.LÀ1, Petronas Ammonium sulphate (10 g.LÀ1, Sodium acetate (4.1 g.LÀ1, Petronas c6.53 Angel Yeast Co. Ltd.) Chemical Company BhD.) Petronas Chemical Company Bhd.) Chemical Company Bhd.) and d7.02 bGM-2 Yeast extract (15 g.LÀ1, Urea (40 g.LÀ1, Merck, Shd. Ammonium sulphate (10 g.LÀ1, Merck, Sodium acetate (4.1 g.LÀ1, Merck, c5.93 Oxoid TM) Bhd.) Shd. Bhd.) Shd. Bhd.) and d6.36

a Technical-grade reagents are not entirely of high purity but are commercially sold for industrial purposes. b Analytical-grade reagents are of high purity and suitable for numerous laboratory applications. c Cultivation media prepared in deionized water. d Cultivation media prepared in tap water.

Table 2 of urease activity is determined as the amount of enzyme which Cost analysis of the ingredients used in this study for bacterial cultivation. degrades 1 mM urea.minÀ1 at 25 ± 2 °C [37]. Medium component *Cost (US$/1kg) *Cost (US$/1kg) for GM-1 for GM-2 2.4. Biomineralization test Yeast extract 18.67 180.00 Urea 1.03 111.25 Ammonium sulphate 3.80 176.90 For the biomineralization test (Fig. 1), two cementation solu- Sodium acetate 4.15 19.51 tions were prepared each containing technical-grade and Total 27.65 487.66 analytical-grade cementation reagents, respectively. The major dif- * The reagents were procured in Malaysia (the exchange rate is MYR 4.11 = USD$ ference between technical-grade and analytical-grade cementation 1.0). reagents is purity. Hence, biomineralization test was essential to evaluate how the purity level affects the biomineralization process when ureolytic bacteria are inoculated into these two cementation Table 3 solutions. The physicochemical properties of the commercially Physiochemical properties of tap water used in this study. available technical-grade cementation reagents are shown in the

Parameter Unit Result Supplementary materials, Table OA1. Technical-grade cementation solution contained 15 g.LÀ1 of food-grade yeast extract (Angel Dissolved oxygen mg.LÀ1 6.42 Biochemical oxygen demand mg.LÀ1 1.90 Yeast Co. Ltd.), 1 M of urea (Petronas Chemical Company Bhd.) À1 Chemical oxygen demand mg O2.L 38.7 and 1 M of calcium chloride (Lianyungang Longyi Industry Co. Total suspended solids mg.LÀ1 1.00 Ltd). The analytical-grade cementation solution contained À1 Total dissolved solids mg.L 51.20 laboratory-grade yeast extract (15 g.LÀ1, OxoidTM), urea (1 M, Mer- Salinity % 0.20 ck, Shd. Bhd.), calcium chloride (1 M, Merck, Shd. Bhd.). Both solu- pH – 7.42 Chloride mg.LÀ1 5.10 tions were prepared by first autoclaving the yeast extract placed in Fluoride mg.LÀ1 0.01 Schott bottles containing 1 L tap water. After cooling, urea and cal- À Sodium mg.L 1 3.46 cium chloride were added and carefully shaken until the reagents À1 Magnesium mg.L 1.05 were totally dissolved. The cementation solutions were further Iron mg.LÀ1 0.01 Aluminium mg.LÀ1 3.83 sterilized using ultraviolet radiation in a biological safety cabinet Nitrate mg.LÀ1 1.26 (Escoglobal) before transferring them into sterile beakers (Fig. 1). Ammonium nitrogen mg.LÀ1 0.01 5 mL of overnight S. pasteurii cultures in GM-1 and GM-2 were inoculated into beakers containing 95 mL of the cementation solutions and incubated aerobically for 72 h at 32 °C with no shaking. At the end of the incubation period, the precipitates were collected 2.3. Urease activity in a centrifuge tube (50 mL) as described by Omoregie et al. [35], oven-dried at 60 °C for 4 h and kept at room temperature Urease activity (mM urea hydrolysed.minÀ1) of the bacterial (26 ± 2 °C) for further analysis. cultures was measured at the end of the incubation period using conductivity measurement method [13]. Urease hydrolyses the urea substrate in the solution to produce ammonium and carbon- 2.5. XRD analysis ate ions and increases the conductivity under standard conditions. Whiffin [13] determined that urease activity could be measured by X-ray diffraction (XRD) is a rapid analytical technique regularly assessing the electrical conductivity changes per minute of the used to determine the mineralogical composition and crystalline bacterial-urea solution. For the conductivity measurement, 10 mL phase of the CaCO3 precipitates after the biomineralization test. of bacterial culture was mixed with 90 mL of urea solution XRD analysis was performed by Quasi-S Sdn. Bhd. Malaysia. Pow- (1.5 M) and the relative conductivity change was measured for dered samples were arranged on a carbon tape and covered with 5 min at 25 ± 2 °C. The gradient, also known as the conductivity gold via sputter-coating prior to microscopic analysis. A Panalytical À À variation rate (mS.cm 1.min 1), was obtained from the plotted system (X’Pert3 Powder) with Cu Ka tube anode having 40 KV and graph slope, multiplied by the dilution factor (10) and converted 35 mA running parameters was used to scan the fine-powdered to urease activity. The urease activity was calculated by taking into samples. The angle of diffraction (2h) of the X-ray was set from account the dilution to provide the undiluted value [36]. Specific 5° to 80°, with a step size of 0.05° and a scanning speed of 5° per À urease activity (mM urea hydrolysed.min 1.OD-1) was determined minute. The analyzed components were identified by comparing by dividing the obtained urease activity by the biomass value them with standards established by the International Centre for (optical density) [13]. In the measured range of activities, one unit Diffraction Data. 4 A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828

Fig. 1. The two cementation solutions containing technical-grade (A) and analytical-grade (B) reagents before being inoculated with S. pasteurii.

2.6. Biocement treatment For comparison purposes, a total of four treatment groups (i.e. different concentrations) were formed and each group was sub- Sandy soil was used for biocement experiments presented grouped into two: a) AG (DW) AG (TW); and b) TG (DW) and TG herein. Particle sizes of the sand ranged from fine sand (TW). For each cementation solution used, the urea and calcium (0.075 mm) to fine gravel (4.75 mm) and are classified as poorly chloride were added into Schott bottles containing sterilized 5 g. graded in accordance with Unified Soil Classification System LÀ1 of yeast extract. The cementation solutions were further sub- (USCS). Key properties of the poorly graded soil were 0.22 mm, jected to UV-sterilization before carrying out the sand treatment.

0.27 mm, 0.35 mm, 1.60 and 0.95 for D10,D30,D60,Cu and Cc, The ureolytic bacterial cultures used for the biocement treatment respectively. The dry density and pH value of the sand were were cultivated in GM-1 and GM-2 prepared in deionized water 1.67 g/cm3 and 7.10, respectively. Polyvinyl chloride (PVC) tubing and tap water. The cultures were added to sand samples which with an internal length of 11.8 cm in length and diameter of were to be treated with corresponding reagents. For example, a 5.8 cm served as a column in this experiment. The PVC tubes were sand column containing S. pasteurii cultivated in GM-1 (tap water) careful vertically cut into halves and tightly held together with will likewise be treated with technical-grade cementation solution plastic seal strips. The columns were then placed on polypropylene prepared in tap water. To achieve cementation, surface percolation sheets and the spot where the columns were to be placed was per- was used to introduce cultures and cementation solutions. All the forated with holes to allow discharge of effluent during treatment. solutions were applied from the top of the columns and allowed to The holes were covered with WhatmanÒ cellulose filter papers to slowly percolate by gravity and capillary forces [37]. act as filters before the columns were then placed upon the filter The sand columns were treated with 195 mL of bacterial cul- papers. 350 g of dry sterile sand was placed into each column tures and cementation solutions in triplicate. Columns were first before being subjected to biocement treatment (Fig. 2). To enable percolated with bacterial cultures and allowed to settle for 6 h the collection of effluents during treatment, a plastic container before the cementation solutions were added. The treatment was was placed below the polypropylene sheets in a fume hood (Lab repeated for three more days and reaction time was 24 h. For each Craft Basix). day of biocement treatment upon the soil samples, the bacterial The following acronyms AG (DW), AG (TW), TG (DW) and TG cultures were added prior to each application of cementation solu- (TW) represented analytical-grade deionized water, analytical- tion. The treatment was performed at room temperature grade tap water, technical-grade deionized water and technical- (26 ± 2 °C). After the treatments, 30 mL of deionized water and grade tap water, respectively. The sand columns were treated using tap water were respectively used to flush the sand columns to four different concentrations of cementation solutions of 0.25 M, wash out any soluble salts which might have formed as by- 0.50 M, 0.75 M and 1.0 M. Equal molar of urea and calcium chloride products [3]. The biocemented sand samples were carefully dis- concentrations were prepared in deionized water and tap water. mantled from their respective columns and allowed to cure for

Fig. 2. Setup of sand columns prior to biocement treatment. A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 5

3 weeks at room temperature. After completion of the curing per- with accelerating voltage of 15 kV, magnification 5Â up to iod, each sample was subjected to a surface strenght test, CaCO3 300,000Â and resolution of 3.0–4.0 nm. The crushed samples were content test and SEM analysis. For pH analysis of effluents from properly arranged on carbon tape, sputter-coated with a thin layer samples during biocement treatment, 20 mL of the effluents from of gold and placed onto the stub before being analysed. EDX spec- the columns were extracted and retained for further analysis. The troscopy was used examine the elemental compositions of the effluents were placed in sterile plastic centrifuge tubes (50 mL technical-grade and analytical grade reagents (yeast, urea and cal- capacity) and their respective pH were examined for detection of cium chloride) used in this study. urease activity after treatment. The initial pH of all cementation solutions was measured before the soil biocement treatment (Sup- 2.10. Statistical analysis plementary materials, Table OA2) for comparison purpose. All the experiments in this work were carried out in triplicate. 2.7. EDXRF analysis The experimental results were analysed using the statistical analysis software GraphPad PrismÒ (version 8) to determine the signif- Energy dispersive X-ray fluorescence (ED-XRF) spectrometry is icance of differences between the means of groups obtained in this a non-destructive analytical technique used to analyse and quan- study. One-way analysis of variance (ANOVA) and Tukey-Kramer tify the relative concentration of samples by measuring the wave- post hoc test were performed using MinitabÒ (version 18). The sig- length and intensity generated through fluorescent X-rays. The nificance level was set at P < 0.05. analysis was performed by Quasi-S Sdn. Bhd. Malaysia using an X-ray fluorescence spectrometer (Thermo Electron /Model ARL 3. Results Quant’X). ED-XRF analysis was only performed on technical- grade cementation reagents (yeast extract, urea and calcium chlo- 3.1. Growth performance of S. pasteurii ride) to verify the elements and concentration of each reagent since they are not of highest purity grades. ED-XRF spectrometry The growth profiles of S. pasteurii after being incubated aerobi- was used to analyse the samples because it is cost-effective, fast cally for 72 h at 32 °C under shaking conditions (130 rpm) in differ- and provides accurate results. X-ray fluorescence analysis was per- ent cultivation media (Supplementary materials, Fig. OA1) which formed using the following parameters; filter (cellulose), voltage were prepared using deionized water and tap water are presented (7 kV), Maximum Energy (40 keV), emission current (1 mA), power in Fig. 3A. Cultivation of the bacteria from single colonies showed it (1 mA) and lifetime (50 s). The collected dried samples were placed could grow well in GM-1 similarly to GM-2. Following a lag phase into the sample holder section of the spectrophotometer. Bulk of 6 h, the bacteria displayed logarithmic phase growth in all media analysis of the sample was performed under vacuum and the X- ray fluorescence data were obtained. GM-1 (Deionized Water) GM-1 (Tap Water) 2.8. Surface strength and CaCO3 content GM-2 (Deionized Water) GM-2 (Tap Water) A simple, lightweight hand-held pocket penetrometer (ELE A International, 38-2695) was used to measure the strength of the 1.6 treated sand samples. The pocket penetrometer is commonly used ) in geotechnical engineering to rapidly estimate the surface 600 1.2 strength of cohesive soils after biocement treatment [37,38]. The penetration piston (6.4 mm) was manually pushed into the treated soil until the penetration resistance value from the calibrated 0.8 spring was obtained on the scale values engraved onto the penetrometer barrel. The penetration piston was pushed from the top section of all treated sand samples. The penetrometer used in 0.4 this study had a scale which ranged from 0 to 4826 kPa [32]. After a strength test of each biocemented samples, CaCO content was Optical density (OD 3 0.0 measured using the gravimetric acid washing technique [39]. Por- 0 122436486072 ° tions (100 g) of the treated samples were oven-dried at 60 C for Incubation time (h) 24 h before subjecting to acid wash. The samples were weighed before and after being digested in 1 M hydrochloride acid under B 9.5 continuous stirring [3]. The dissolved CaCO3-acid washed solutions were rinsed several times, filtered using WhatmanÒ cellulose filter 9.0 paper, allowing the dissolved CaCO3 to be rinsed off while retaining the sand grains. The difference in the two mass of the sand before 8.5 and after acid wash was determined [39]. The CaCO3 content (%) pH was expressed as the ratio of CaCO3 weighed prior and after acid 8.0 wash. 7.5 2.9. SEM-EDX analysis

7.0 The surface topography, morphology and mineralogical compo- 0 122436486072 sitions of samples subjected to biocement treatment with 1 M Incubation time (h) cementation solution were investigated at the Biotechnology Research Institute, Universiti Malaysia, Sabah using scanning elec- Fig. 3. Monitored (A) growth and (B) pH profiles of S. pasteurii strain when tron microscope (SEM). SEM micrographs were obtained using cultivated in Growth Medium-1 (GM-1) and Growth Medium-2 (GM-2). The Hitachi S-3400 N attached with energy dispersive X-ray (EDX) unit, vertical error bars represent mean ± SD (n = 3). 6 A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 until approximately 30 h of incubation period. The cultures precipitations occurred. The precipitates formed in the technical- remained at stationary phase until the end of the cultivation period grade cementation solution and quickly settled at the bottom of (72 h). At the end of the experiment, the maximum OD for the ure- the conical flask after approximately 1 h of ureolysis reaction per- olytic bacteria all the growth medium was 1.2 ± 0.01. The kinetics iod while the precipitates formed in the analytical-grade cementa- parameters which includes the growth rate and doubling time tion solution moderately settled at the bottom of the conical flask for the cultivation medium used to study the performance of after approximately 4 h of ureolysis reaction period. S. pasteurii can be seen in Table 4. After the biomineralization test, the collected precipitate sam- Since alkalinity is one of the essential factors of MICP, pH ples (Supplementary materials, Fig. OA2) were oven-dried before profiles of ureolytic bacteria in the cultivation medium were also characterization by XRD analysis to identify the precipitates. XRD determined. In Fig. 3B, it was observed that the pH profiles were analysis provides direct qualitative and quantitative evidence of similar to growth profiles of the ureolytic bacteria. As bacterial cul- CaCO3 precipitation from MICP by determining the number of dif- ture densities increased during incubation, the pH values increased ferent phases in the selected samples [40]. The XRD analysis simultaneously. Immediately after inoculation of the bacterial star- (Fig. 5) showed that the precipitates induced by S. pasteurii in ter cultures, the pH values increased from 7.0 (initial pH medium) analytical-grade and technical-grade cementation solutions were to 7.5 ± 0.01–7.9 ± 0.02, serving as an indication of urea hydrolysis. identified as CaCO3 having almost undistinguishable polymorphs At the end of the incubation period, the maximum pH of all culture peaks. The precipitates obtained from analytical-grade solution medium were between 9.2 ± 0.01–9.3 ± 0.01. Following the proce- produced calcite (93%) and vaterite (7%), while that of technical- dures described by Whiffin [13], the urease activities of S. pasteurii grade solution produced calcite (92.6%) and vaterite (7.4%). cultures were determined by measuring their electrical conductiv- ities at the end of the incubation period. The maximum urease activity and specific urease activity were 21.1 ± 0.06 mM urea 3.3. Soil biocementation, ED-XRF and EDX analyses hydrolysed.minÀ1 and 18.6 ± 0.10 mM urea hydrolysed.minÀ1. À OD 1 from GM-2 (tap water), while the lowest urease activity Four different concentrations of cementation solutions ranging À (17.4 ± 0.19 mM urea hydrolysed.min 1) and specific urease activ- from 0.5 to 1.0 M were used in the current study. The sand samples À À ity (14.9 ± 0.19 mM urea hydrolysed.min 1.OD 1) were found in which were treated with ureolytic bacterial cultures and cementa- GM-1 (deionized water). tion solution (1 M) through surface percolation are presented in Supplementary materials, Fig. OA3. Surface percolation was used 3.2. Biomineralization and XRD analysis in his experiment due to its ease of operation. This method does not require heavy machinery and expensive treatment techniques For biomineralization test of cementation solutions prepared [41]. However, it is limited with treatment depth as it is most suit- using technical-grade and analytical-grade reagents in tap water able for solution penetration into the soil to a depth of 2 m [42]. (Supplementary materials, Fig. OA1), ureolytic bacteria previously Hence, the use of the surface percolation method was suitable for cultivated in GM-1 and GM-2 were inoculated, respectively into this current study since the depth of the PVC columns was less these solutions. Upon addition of the bacterial cultures, slurry pre- than 1 m. The use of technical-grade cementation solution for soil cipitations (Fig. 4) formed almost immediately. There were notice- biocalcification has an enormous economic advantage over able visual differences in the rate at which the insoluble analytical-grade solution.

Table 4 Growth kinetics of S. pasteurii NB28 (SUTS) in different cultivation media.

Kinetics parameters GM-1 (deionized water) GM-1 (tap water) GM-2 (deionized water) GM-2 (tap water) Speciﬁc growth rate (hÀ1) 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 Doubling time (h) 18.19 ± 0.08 17.28 ± 0.21 18.95 ± 0.12 18.02 ± 0.20

Minimum OD600 0.1 ± 0.00 0.1 ± 0.00 0.1 ± 0.00 0.1 ± 0.00

Maximum OD600 1.2 ± 0.01 1.2 ± 0.01 1.2 ± 0.00 1.2 ± 0.01 Minimum pH 7.6 ± 0.02 7.9 ± 0.02 7.7 ± 0.04 7.5 ± 0.01 Maximum pH 9.2 ± 0.01 9.3 ± 0.01 9.2 ± 0.01 9.3 ± 0.01 Urease activity (mM urea hydrolysed.minÀ1) 17.4 ± 0.19 19.2 ± 0.03 19.1 ± 0.75 21.1 ± 0.06 Speciﬁc urease activity (mM urea hydrolysed.minÀ1.OD-1) 14.9 ± 0.19 16.6 ± 0.05 16.3 ± 0.72 18.6 ± 0.10

The data are given as mean ± SD (n = 3).

Fig. 4. Ureolysis-driven CaCO3 formation in beakers containing (A) technical-grade cementation solution and solution (B) analytical-grade cementation solution after addition of S. pasteurii cultures. A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 7

Prior to biocement treatment, the pH of each cementation solu- AG (TW) were in the range of 7.44 ± 0.03–9.31 ± 0.02 and tion used was measured as shown in Supplementary materials, 7.39 ± 0.01–9.16 ± 0.01, respectively. Additionally, the pH Table OA2, indicating the solutions were acidic. The initial pH of effluents for TG (DW) and TG (TW) were in the ranges of AG (DW) ranged between 5.05 ± 0.04–5.42 ± 0.12, while AG (TW) 7.44 ± 0.05–9.26 ± 0.01 and 7.67 ± 0.12–9.26 ± 0.02, respectively. ranged between 5.23 ± 0.02–5.40 ± 0.03 for the cementation solu- The ED-XRF analysis (Fig. 7A) carried out on yeast extract, a tions (0.25–1.0 M). On the other hand, the initial pH of TG (DW) food-grade material commonly used for baking and cooking pur- ranged between 4.36 ± 0.03–4.53 ± 0.03, while AG (TW) ranged poses, showed that phosphorus (3.73%), sulphur (3.84%), chlorine between 4.58 ± 0.05–4.88 ± 0.03 for the cementation solutions (43.03%) and potassium (49.40%) were present. The ED-XRF (0.25–1.0 M). The results for pH effluents in Fig. 6 illustrated fluc- analysis (Fig. 7B) of calcium chloride, widely used industrially as tuating pH values in different concentrations for all the samples a de-icing agent on winter roads, showed that sodium (13.70%), treated with analytical-grade and technical-grade cementation magnesium (0.65%), calcium (30.77%), chlorine (54.64%) and sul- solutions. In all the collected effluent samples (0.25–1.0 M), it phur (0.243%) were present. The ED-XRF analysis (Fig. 7C) was also was observed that the measured pH for AG (DW) and performed on urea, widely used in the agriculture industry as a

Fig. 5. XRD pattern of CaCO3 polymorphs obtained from (A) analytical-grade and (B) technical-grade samples. 8 A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828

0.25M B 0.50M A 9.5 10.0 AG (DW) AG (DW) 9.0 AG (TW) 9.5 AG (TW) TG (DW) TG (DW) 8.5 TG (TW) 9.0 TG (TW)

8.0 8.5 pH of effluents pH of effluents

7.5 8.0 24 48 72 96 24 48 72 96 Incubation time (h) Incubation time (h)

0.75M 1.0M C 9.5 D 9.0 AG (DW) AG (DW) 9.0 AG (TW) 8.5 AG (TW) TG (DW) TG (DW) 8.5 TG (TW) 8.0 TG (TW)

pH of effluents 8.0 7.5 pH of effluents

7.5 7.0 24 48 72 96 24 48 72 96 Incubation time (h) Incubation time (h)

Fig. 6. pH of the efﬂuents collected from treated sand columns. The vertical error bars represent mean ± SD (n = 3). nitrogen fertilizer for soil nourishment. The spectrophotometer It can be seen that the results of treated samples with high-grade could only detect the presence of calcium (82.90%) and chloride and low-grade cementation solutions were comparable. The result (17.10%) in the samples. showed that for the sample group treated with cementation For a comparative analysis of the technical-grade and analytical reagents at a concentration of 0.25 M, AG (TW) had highest surface grade reagents used in this study, EDX was used to determine ele- strength (1793.00 ± 138 kPa) while TG (DW) had lowest surface mental compositions and concentrations of the reagents (Table 5). strength (1448.00 ± 69.00 kPa). For the samples which were trea- Based on the analysis for analytical-grade and technical-grade ted with cementation reagents at a concentration of 0.50 M, TG yeast extract media, carbon had the highest values (51.44 ± 1.89% (DW) showed the highest strength (3861.00 ± 365.11 kPa) while and 46.51 ± 1.53%) among all the elements detected, while sodium AG (TW) had the lowest strength (2941.67 ± 443.04 kPa). Addition- (0.18 ± 0.03%) and sulphur (0.29 ± 0.06%) had the lowest values for ally, when the sand samples were treated with cementation the high-purity and low-purity reagents, respectively. Additionally, reagents at a concentration of 0.75 M, AG (DW) had the highest a trace amount of aluminium, (0.43 ± 0.07%) was detected only in strength (4734.34 ± 158.77 kPa) while TG (TW) had the lowest technical-grade yeast extract media. The EDX analysis for strength (4274.67 ± 497.01 kPa). Contrary to the rest of the groups, analytical-grade and technical-grade urea showed that nitrogen when the sand columns were treated with 1.0 M of cementation had the highest values (38.62 ± 0.48% ad 39.74 ± 0.16%) among all solutions, they all had same strength values (4826.00 ± 00 kPa). the elements detected, while calcium had the lowest values Statistical analysis using one-way ANOVA showed that there were (0.01 ± 0.00% and calcium (0.32 ± 0.42%) for the high-purity and signiﬁcant differences (P-value is 1.63E-19) among the group low-purity reagents. The EDX analysis for analytical-grade and means of the analysed results. technical-grade calcium chloride showed that chlorine had the highest values (51.5 ± 2.11% and (47.80 ± 1.44%) among all the ele- 3.5. CaCO3 contents of the biocemented sand samples ments detected, while oxygen (10.84 ± 1.08%) and magnesium (0.97 ± 0.48%) both had the lowest values. Additionally, sodium CaCO3 content measurements are often performed to determine (1.36 ± 0.31%) alongside magnesium were only detected in the increases in CaCO3 following biocement treatment of soil samples technical-grade reagent. [14]. Fig. 9 shows the CaCO3 content of the biocemented sand specimens treated with ureolytic bacterial cultures and different con- 3.4. Surface strength of the biocemented sand samples centrations of analytical-grade and technical-grade cementation reagents. The sample group treated with cementation reagents at

Fig. 8 shows surface strength of the biocemented samples after a concentration of 0.25 M, AG (DW) had the highest CaCO3 content treatment with ureolytic bacterial cultures and different concen- (7.1 ± 1.72%) while TG (DW) had the least CaCO3 content trations (0.25–1.0 M) of analytical-grade and technical-grade (5.6 ± 1.15%). For the samples which were treated with cementa- cementation reagents in deionized water and tap water for 96 h. tion reagents at a concentration of 0.50 M, TG (TW) had the highest A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 9

Table 5 Comparison of technical-grade and analytical-grade elemental compositions.

Grade type Cementation reagents and elemental compositions (atomic %) Yeast extract Urea Calcium chloride Analytical- carbon carbon carbon (4.6 ± 0.27), grade (51.4 ± 1.89), (23.9 ± 0.75), nitrogen nitrogen nitrogen (11.0 ± 1.35), (7.4 ± 0.38), oxygen (38.6 ± 0.48), oxygen (32.0 ± 1.70), oxygen (10.8 ± 1.08), sodium (0.2 ± 0.03), (34.5 ± 0.74), chlorine phosphorous aluminium, (51.5 ± 2.11) and (1.9 ± 0.33), (2.8 ± 0.19), calcium sulphur chlorine (22.5 ± 0.51). (1.2 ± 0.11), (0.1 ± 0.02) and chlorine (0.2 ± 0.11) calcium and potassium (0.1 ± 0.00). (5.7 ± 1.24). Technical- carbon carbon carbon (8.6 ± 0.69), grade (46.5 ± 1.53), (21.8 ± 0.69), nitrogen nitrogen nitrogen (7.6 ± 1.20), oxygen (8.9 ± 0.50), oxygen (39.7 ± 0.16), (13.7 ± 1.89), (33.6 ± 2.49), oxygen sodium (1.4 ± 0.31), sodium (5.3 ± 0.73), (37.4 ± 1.01), magnesium aluminium, aluminium, (0.9 ± 0.48), (0.4 ± 0.07), (0.5 ± 0.09), chlorine phosphorous chlorine (47.8 ± 1.44) and (0.8 ± 0.09), sulphur (0.3 ± 0.02) and calcium (0.3 ± 0.06), calcium (22.0 ± 0.93). chlorine (2.7 ± 0.41) (0.3 ± 0.42). and potassium (1.4 ± 0.14).

The data are given as mean ± SD (n = 3).

AG (DW) TG (DW) AG (TW) TG (TW)

a ab ab 5000 abc a a a a cde bcd 4000 e de 3000

f 2000 f f f

1000

Surface strength (kPa) 0

0 M 0M .5 75 M 1. 0.25 M 0 0. Cementation solutions (w/v)

Fig. 8. Surface strength measurement of the biocemented sand samples using pocket penetrometer. Treatments were carried out using ureolytic bacterial cultures with analytical-grade and technical-grade solutions. The acronyms AG Fig. 7. ED-XRF spectrum results of the commercially available technical-grade (DW), AG (TW), TG (DW) and TG (TW) represent analytical-grade deionized water, reagents used for biocement treatment. (A) yeast extract, (B) calcium chloride and analytical-grade tap water, technical-grade deionized water and technical-grade (C) urea. tap water, respectively. To determine whether the differences between group means are statistically significant at P < 0.05, one-way ANOVA with Tukey-Kramer post hoc analyses were used. The rectangular bars that share a letter (i.e. a, b, c, d, e, f) indicates there are no statistical significant differences between their means. The CaCO3 content (14.7 ± 1.84%) while AG (TW) had the least CaCO3 vertical error bars represent mean ± SD (n = 3). content (12.2 ± 0.47%). Additionally, samples with cementation reagents at a concentration of 0.75 M showed that AG (DW) had the highest CaCO3 content (23.7 ± 1.32%) while TG (TW) had the 3.6. SEM analysis least CaCO3 content (21.7 ± 4.10%). Finally, results from the samples with cementation reagents at a concentration of 1.0 M showed In order to visualise the microscopic structure of the sand col- that TG (TW) had the highest CaCO3 content (33.2 ± 0.59%) while umns after biocement treatment, SEM analysis was performed AG (TW) had the least CaCO3 content (31.2 ± 1.97%). Statistical and the samples treated with 1 M cementation solutions were analysis showed that there were significant differences (P-value compared with an untreated sand sample. The SEM image in Sup- is 0.0) among the group means. plementary materials, Fig. OA5 clearly illustrates the absence of 10 A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828

AG (DW) TG (DW) be absorbed by the microorganism for quicker growth. This could be the reason there were noticeable differences in the growth pat- AG (TW) TG (TW) terns of the bacteria in the growth medium (Fig. 3A). The pH pro- 35 a a a files (Fig. 3B) of all cultivation medium increased steadily until the a end of the incubation period. The escalation of pH was due to urea 30 hydrolysis by microbial urease which is essential for CaCO3 precip- b b 25 b b itation [43]. Ureolysis allows the changes of pH to alkaline in the presence of 20 ammonia and carbonate ions. From 36 h until the end of incuba- c tion, the pH values stabilized which could be a result of depletion contents (%) c c 15 c 3 in biomass production and urease activity. The hydrolysis of urea 10 d d by urease occurs more at a higher bacterial concentration which, d d in turn, leads to high pH values but when bacterial concentration CaCO 5 is low, pH of the culture medium correspondingly declines [44]. 0 The cultivation of ureolytic bacteria in deionized water and tap 0.25M 0.50M 0 .75M 1.0M water was performed to determine if the presence or absence of inorganic materials in water would influence the bacterial growth Cementation solutions (w/v) performance or urease activity. Typically, tap water is treated through water purification systems prior to removal of elements Fig. 9. CaCO3 contents of the biocemented sand samples. Treatments were carried out using ureolytic bacterial cultures with analytical-grade and technical-grade or heavy metals. However, the results in Fig. 3 suggested S. pas- solutions. The acronyms AG (DW), AG (TW), TG (DW) and TG (TW) represent teurii was not noticeably affected when cultivated in growth med- analytical-grade deionized water, analytical-grade tap water, technical-grade ium prepared tap water. Tap water can be used for biocalcification deionized water and technical-grade tap water, respectively. To determine whether because the concentration of nutrients or cementation reagents the differences between group means are statistically significant at P < 0.05, one- way ANOVA with Tukey-Kramer post hoc analyses were used. The rectangular bars exceeds the concentration of inorganics present in tap water by that share a letter (i.e. a, b, c, d, e, f) indicates there are no statistical significant one or two orders of magnitude [25]. However, it was observed differences between their means. The vertical error bars represent mean ± SD that S. pasteurii had higher biomass production when cultivated (n = 3). in GM-1 and GM-2 prepared with deionized water. On the other hand, the urease activities were higher when S. pasteurii was cultivated in GM-1 and GM-2 prepared with tap water. Deionized water crystals in the untreated sand while Fig. 10 shows abundant crystal is commonly used for the preparation of cultivation medium to formation on surface of the treated soil particles. The crystal mor- avoid the influence of contaminating ions during bacterial growth phology of CaCO3 precipitates showed large abundant spheroidal [17], but it seemed the use of tap water favoured the urease activ- shapes with rough surfaces. The SEM observations suggested there ity of S. pasteurii, probably due to the presence of trace elements. are no significant differences among the morphological structures This might be influenced by the electronegativity of bacterial cell of the CaCO3 precipitates formed on the surface of the treated sand walls, thus allowing adsorption of cations from the trace elements particles. The high magnification SEM images in Fig. 11 clearly and enhancing urease activity [43]. show different topographical features of CaCO3 crystal precipitates The ability of S. pasteurii to grow and produce high urease activ- formed on the sand particles when treated with different cementa- ity in GM-1 suggests that this low-cost medium could serve as a tion solution. Fig. 11A shows detailed flower-like spherical struc- replacement substrate to GM-2 which is prohibitively expensive. tures in analytical-grade cementation solution (1 M) prepared in The reason the ureolytic bacterial growth in GM-1 was similar to deionized water. The topography of the crystal structures indicates that in GM-2 might be because of the nutrient components in bundles of cubes clustered together. In Fig. 11B, SEM results for GM-1. The food-grade medium comprises yeast extract (50%), mal- crystal treated with technical-grade cementation solution (1 M) todextrin (40%), monosodium glutamate (8.8%) and disodium 50- prepared in deionized water illustrate well-projected fibre-like ribonucleotide (1.2%). The high concentrations of yeast extract hexagonal structures. The topography of the crystal seems like and maltodextrin provide proteins and carbon sources needed for the structures bundle of fibre are tangled together to form a block. bacterial growth, since S. pasteurii is known to grow well in Fig. 11C shows rhombohedral-like structures in crystals formed protein-rich or carbon-rich culture media [13,42]. Although mal- after treatment with analytical-grade cementation solution (1 M) todextrins are commonly used in a wide spectrum of food prod- prepared in tap water. The topography of this crystal suggests ucts, such and nutritional beverages, they have also been the structures are assembled to form an irregular ball shape. reported as suitable alternative carbon and energy sources for ure- Fig. 11D shows blurry, wrinkled-like crystal structures with olytic bacteria (i.e. Bacillus subtilis) [45,46]. Interestingly, the trace technical-grade cementation solution (1 M) prepared in tap water. amount of monosodium glutamate and disodium 50-ribonucleotide In contrast to other SEM images shown in Fig. 11A–C, the topogra- in the technical-grade yeast extract, which serves as a flavour phy of the crystals shown in Fig. 11D does not display vivid fea- enhancer for the food-grade medium, did not inhibit biomass pro- tures, but instead the presence small cubic crystals clustered duction and urease activity of S. pasteurii. together.

4.2. Biomineralization 4. Discussion The precipitation (Fig. 4) which rapidly occurred simultane- 4.1. Bacterial growth in GM-1 and GM-2 ously in both cementation solutions after addition of the bacterial culture showed a strong depletion rate up to 4 h until no more pre- The observed results showed that all S. pasteurii cultures had cipitates formed. The visual observation noticed during the similar biomass, growth rates, doubling time, pH profiles and ureolysis-driven reaction period corresponded with findings urease activities (Table 4). Nutritive molecules in culture media reported in the literature [3,24,47]. Lu et al. [24] reported that often differ and can influences the growth of microorganism [30]. when calcareous sand samples were dissolved into acetic acid, fast Typically, nutrients with small peptides and amino acids will easily occurring carbonate precipitates were observed in the first half A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 11

(A) (B) sand

sand

CaCO3

sand

Fig. 10. SEM analysis showing crystal morphologies of CaCO3 on surfaces of the sand samples treated with S. pasteurii and 1.0 M of different cementation solutions. Analytical-grade (A) and technical-grade (B) reagents in deionized water; analytical-grade (C) and technical-grade (D) reagents in tap water.

hour and quickly showed a decline in the rate of precipitation. In subsequently triggers greater CaCO3 content [12]. The additional another study, Joshi et al. [47] observed significant delay in precip- ingredients such as salts (i.e. monosodium glutamate) and mal- itate formation during the paste mixture period in nutrient broth todextrin present in the technical-grade nutrient media used in paste mix which influenced the setting characteristic of cement this current study might have influenced the reaction rate of CaCO3 when compared with other paste mixtures (i.e. corn steep liquor). precipitation. Well investigated factors such as chemical reagents, However, in one of the corn steep liquor paste mix samples, an biomass concentration, the composition of nutrient, pH, salinity increase setting or reaction time was observed. It was suggested and reaction medium are known to influence CaCO3 precipitation that the presence of high carbohydrate content might have and polymorphs [12,48–50]. Other synthesis factors such as reac- resulted in inhibition of the calcification process. In a recent study tion time, stirring rate and impurities (e.g., Mg) are also known by Phua et al. [3], carbonate precipitates were obtained by dissolv- to influence the formation of CaCO3 polymorphs by influencing ing powdered chalk in lactic acid via an enzyme-induced carbonate the dissolution and recrystallization of crystals [51]. precipitation process with Jack Bean urease. Rapid crystal formation was observed within the first 10 min after addition of the 4.3. XRD analysis reagent into the CaCl2-solution and significantly slowed after 4 h. Ureolytic bacteria play two key roles in the biomineralization The XRD spectra showed that the maximum peak intensities test, first to generate urease for urea hydrolysis which then pro- were clearly observed at 29.4 2h for calcite. Based on the result duces a suitable alkaline environment for carbonate precipitation illustrated in Fig. 5, the values for analytical-grade and technical- and, second, to provide nucleation sites essential for the formation grade samples are identical within the margin of error (92.6 and of carbonate crystallization, morphology and yield of the precipita- 7.4 is identical to 93 and 7). The XRD spectra showed that the tion [44]. Typically, urea and calcium chloride are the two common intensity peak values for the identified calcite obtained from the chemical reagents used for the biomineralization test but nutrients analytical-grade cementation solution and technical-grade cemen- such as yeast extract are often incorporated into cementation solu- tation solution were 1400 and 1280, respectively. Several studies tions to enhance biomass production and urease activity which have also reported identifying calcite as the major CaCO3 12 A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828

Fig. 11. SEM analysis showing the surface topography of CaCO3 after soil biocementation was performed with S. pasteurii and 1.0 M of different cementation solutions. Analytical-grade (A) and technical-grade (B) reagents in deionized water; analytical-grade (C) and technical-grade (D) reagents in tap water.

polymorphs with a trace amount of vaterite [17,28,52,53]. CaCO3 4.4. MICP treatment of sand columns has three natural polymorphs (i.e. calcite, aragonite and vaterite); calcite is the most thermodynamic stable form under the ambient MICP is often regulated by the concentration of ions, concentra- conditions [51]. In our previous studies [35], XRD analysis of pre- tion of organic carbon, pH of the microenvironment and availabil- cipitated samples obtained after biomineralization test was per- ity of nucleation site [43]. Therefore, the effect of various formed in analytical-grade and technical-grade cementation conditions (i.e. cultivation medium, concentration of cementation solutions both prepared in deionized water showed that the pre- reagents, water source for cementation solution, surface percola- cipitates from analytical-grade solutions were identified as calcite tion treatment method) on soil biocalcification were investigated. (100%). On the other hand, technical-grade cementation solution In addition, a comparison was carried out to determine if was identified as calcite (94.9%) and vaterite (5.1%) polymorphs. technical-grade cementation reagents of different concentrations Irrespective of whether analytical-grade or technical-grade cemen- can replace the standard analytical-grade reagents. Since these tation solutions were prepared in deionized water or tap water, it low-grade chemicals are inexpensive and less harmful to the appears that when biomineralization is performed under labora- geo-environment than pure chemicals, it was necessary to investi- tory conditions, calcite is mostly formed as the dominant CaCO3 gate their potential feasibility for MICP applications [7]. polymorphs while vaterite is formed in trace amounts. In most During the soil treatment, there were comparable outcomes MICP studies, calcite crystal precipitates are often reported as the from the samples treated with analytical-grade and technical- dominant CaCO3 polymorph and this might be because most bioce- grade cementation solutions. Some of the samples treated with ment treatments are typically performed at ambient conditions. higher solution concentrations (i.e. 0.75 M and 1.0 M) showed Calcite formation is the most direct manifestation of MICP since the formation of precipitates on the surface of the sand. Clogging

CaCO3 is the final product of the process [40]. However, other MICP formation is a common problem during the biocement treat- studies that have reported vaterite and aragonites as the dominant ment when using percolation or injection treatment [42,54,55]. crystals upon XRD analysis of their precipitated samples [29,49]. This is also often influenced by using a higher concentration of A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 13 cementation solutions or when multiple treatments are used for more pronounced fall in pH was observed when compared with soil biocementation. The issue with using multiple cementation the technical-grade samples. This was especially noticeable in solution treatments involves the formation of bio-clogging espe- 0.5 M and 0.75 M samples after the 48 h incubation period. The cially at the site of entry during treatment (Supplementary materi- increase or decrease of pH during MICP is mostly attributed to als, Fig. OA3). This formation inhibits further penetration of urease activity. Hence, the fall of urease activities as the incubation cementation solution into the soil particles and, instead, allows period progressed from 24 to 96 h occurred in all measured sam- accumulation of the CaCO3 on the surface of the soil samples ples. There are several possibilities to explain the observed decline (treatment point of entry). The reaction of the ureolytic bacteria in the urease activities of the effluent samples, including depletion and cementation solution around the treatment point of entry lim- of necessary nutrients, accumulation of ammonia or carbonate its penetration into the soil [41]. Repeated injection or addition of ions, and production of extracellular metabolites [47]. The fall of the bacterial culture and cementation solution eventually leads to pH in the analytical-grade and technical-grade samples might be less CaCO3 formation inside the soil matrix and uneven CaCO3 attributed to the purity of the reagents used for soil biocementa- distribution [42]. Hence, proper treatment interval between the tion. The EDX analysis (Table 5) indicated there were noticeable bacterial culture and cementation solution should be taken into differences in the concentrations of elements present in the consideration [48]. cementation reagents. For example, the technical-grade yeast Surface clogging can also lead to self-enhancement of strength extract needed for microbial growth contained trace amounts of due to the increase in crystal and decreased porosity [42]. During aluminum and sulphur, while technical-grade calcium chloride the treatment, it was noticed that samples treated with 0.75 M showed trace amounts of sodium and magnesium which were and 1.0 M concentrations of the cementation solutions showed absent in the analytical-grade reagents. It was previously sug- decreased soil permeability after 48 h. The reduction of permeabil- gested that the concentration of magnesium has a remarkable ity allows an increase in the hydraulic retention time of the cemen- effect on MICP, CaCO3 deposition rate and polymorphs [62]. Hence, tation solution during infiltration and more crystal formation at it is possible that the presence of these elements (aluminum, sul- the treatment regions [42]. However, it has been highlighted that phur, sodium and magnesium) could affect urease activities and, excess soil clogging of CaCO3 formation at the treatment site can thus, might be the reason for the less pronounced fall in pH result in improper attachment and retention of the bacterial cul- observed for the technical-grade samples. ture in the soil matrix, thus affecting the homogeneity of CaCO3 distribution and strength of the soil [41]. Thus, after the biocement 4.6. Cost analysis test, the strength of the soil and CaCO3 content were analysed (see Sections 3.7 and 3.8). Early microbial activity upon inoculation of The use of technical-grade cementation solution for soil the ureolytic bacterial culture alongside the cementation solution biocalcification has an enormous economic advantage over initiates premature formation of CaCO3 precipitates which clog analytical-grade solution. The calculated cost for the technical- the injection points [38]. This clogging inhibits free flow of the grade cementation reagents of 0.25–1.0 M concentrations ranged cementitious material between soil particles, hence resulting in between 0.07 and 0.26 US$/L which is considerably lower than un-even CaCO3 distribution [56]. Obtaining complete uniformity the cost of for the analytical-grade cementation reagents for the may be very challenging but researchers are exploring ways to same concentrations (3.33–13.29 US$/L). This shows a cost differ- resolve this biocement limitation through different techniques ence of 47 to 51-fold between the analytical-grade solution and such as multiple injection, surface percolation, bioslurry, one- technical-grade based on the market price of the reagents procured phase low-pH injection and temperature reduction [42,56–59]. in Malaysia. The use of the commercially available technical-grade Results from the aforementioned techniques showed that initial reagents was able to tremendously reduce treatment cost, which is clogging formation at the injection points were reduced, allowing a considerable advantage over the costs of standard reagents an improvement in the distribution of the cementation solution, typically used in biocement studies. Utilization of low-cost enhanced strengths of the treated soil samples and homogenous cementation reagents would certainly be suitable for future MICP contents of CaCO3 precipitate formed between the soil particles. field-scale advancements [7]. All the growth media and chemicals prepared in this study were 4.5. pH of effluent samples sterilised by autoclaving before use to ensure control and reproducibility, although this is not viable for large-scale or field-scale pH is a key parameter used to measure urease activity of implementation. The use of sterile equipment is also not practical microorganism during MICP process, hence the pH of effluent sam- or feasible due to the sheer size of the construction machinery. ples was analyzed which ranged between 7.39 ± 0.01–9.26 ± 0.02. Additionally, outsourcing sterilization services from biotechnolog- The results are similar pH of effluent samples previously reported ical companies is possible but this would also drive up the MICP (8.5–9.3) [9,60,61]. It was suggested that the reason pH effluents construction costs. Recognising this challenge, future large-scale varied was due to different bacterial cell concentration. The rise MICP investigations should explore the use of non-sterile reagents in pH values was due to greater urease activities produced by a for bacterial cultivation and biocement treatment. It is understand- higher concentration of ureolytic bacteria [60]. The analysis of able that other microorganisms (urease-producers or non-urease the pH effluents illustrates that urea hydrolysis occurred from producers) might influence the MICP process. In addition, the the first day of biocement treatment until the final treatment on growth of non-desired microbes during bacterial cultivation and the fourth day. This situation was also reported by Dhami et al. treatment using non-sterile reagents could result in competition [9] for effluents studied up to ten days. It was also noted that for nutrients with the introduced microbe (i.e. S. pasteurii) and, throughout this treatment, the pH of the effluents remained alka- thus, affect bacterial growth, urease activity or CaCO3 precipitation. line which indicated active urease activity of the bacteria in all To limit this occurrence, two approaches could be adopted as a samples irrespective of treatment with analytical-grade or low-cost method of preparing growth media solutions and chemi- technical-grade cementation solutions. In Fig. 6, the pH effluent cals, namely, filtration and steam. The potential outcome of per- profiles of samples collected and measured after biocement treat- forming MICP studies under non-sterile conditions might address ment on sand columns using technical-grade and analytical-grade the economic challenges of supplying large-scale volumes of ure- cementation solutions shows depletion of the pH values. For all olytic bacterial cultures for future in situ MICP studies or construc- concentrations (0.25–1.0 M) of analytical-grade samples, a much tion applications. 14 A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828

4.7. Surface strength, CaCO3 contents and SEM analysis However, when comparisons were made between groups, it was observed that there were no correlations between the results from

The concentration of cementation solution is one of the key surface strength and CaCO3 content. For example, in samples trea- factors which affect CaCO3 crystal formation [7]. Hence, sand ted with 0.25 M, AG (TW) had the highest strength value, while AG biocement formation using different solution concentrations was (DW) had the highest CaCO3 contents. For samples treated with assessed with technical-grade reagents compared with analytical- 0.5 M, TG (DW) had the highest strength value, while TG (DW) grade reagents under the same conditions. The results of the had the highest CaCO3 contents. For the sample group treated with penetrometer tests (Fig. 8) showed that the biocement treatment 0.75 M, AG (DW) had the highest values for surface strength and process based on ureolytic bacteria and cementation solutions CaCO3 contents. However, it was noticed that for samples treated was able to improve the surface strength of the sand samples. The with 1.0 M, all AG and TG samples had equal strength values which result also showed that the increase in concentration of cementa- is understandable because of the scale for the penetrometer used tion solutions correlated with increased strength of the treated in this study to measure the surface strength of treated samples. samples since higher amounts of calcium can induce greater CaCO3 The maximum surface strength reading obtained for samples trea- formation and improve soil strength. From an economic point of ted with 1.0 M of cementation solution was 4826.00 ± 00 kPa, view, lower concentrations might be preferred but cementation reaching the maximum level on the scale of the pocket penetrom- treatment needs to be completed rapidly, hence higher concentra- eter. There is a signiﬁcant possibility that the surface strength of tions are recommended [63]. To minimize the cost of using a high these biocemented sand sample would vary if a different concentration of the cementation reagents, the technical-grade penetrometer with measuring scales exceeding the range of reagents will serve as a suitable choice. It is important to note that 4826.00 ± 00 kPa was used. However, CaCO3 contents did not show the cementation reaction might not only be inﬂuenced by reagent similar result as the strength results for this group. Instead, the concentrations; other factors such as temperature, injection speed, CaCO3 contents result showed that TG (TW) had the highest value treatment interval and treatment duration play essential roles for samples treated with 1.0 M.

[40,41,64]. For the biocementation, the samples were treated and SEM analysis (Figs. 10 and 11) of the CaCO3 crystals formed by allowed to cure at room temperature. The treatment solutions were S. pasteurii after soil biocementation showed that with the use of treated once a day for a duration of 92 h. This might have supported technical-grade cementation reagents, similar morphological char- the proper formation of crystals or bonding in the sand matrix espe- acteristics were obtained to that of analytical-grade cementation cially for samples treated with higher concentrations resulting in reagents. CaCO3 crystals with different morphologies in the treated higher compressive strengths. sand samples have been reported in previous MICP studies

The most common unconﬁned compression tests are typically [3,44,49]. The morphological characteristics of CaCO3 crystal can carried out on a compression machine to describe the compressive be inﬂuenced by various conditions such as type of bacterial strength of biocemented soils as reported by several researchers strains, urease activity and nucleation site [43]. Alternatively, [3,22,24,28,30,53,63], but available reports in the literature have microbial extracellular polymeric substances mainly composed of shown that pocket penetrometers can also be used to evaluate polysaccharides, proteins and DNA, are known to improve the the surface (top) strength of treated samples [37,38,42]. Typically, aggregation of soil particles and contributes to production of differ- many local strength points are selected, hence in this study, several ent CaCO3 polymorphs or crystal morphologies [54]. Based on the sets of triplicate samples were prepared and each set contained SEM analysis, it seems technical-grade cementation reagents can three replicates to enhance repeatability of the treatment and sup- serve as suitable alternatives to analytical-grade cementation port the strength test. The improvement or changes of strength reagents since they both precipitate CaCO3 crystals having similar values for the samples differed due to the deposition of CaCO3 by morphological structure. the ureolytic bacteria within the pores of the sand matrix [54].

The formation of CaCO3 crystal and its contents within the sand particles are essential for soil solidification and improvement of 5. Conclusions soil strength. It was also observed that the addition of yeast extract as an ingredient of the cementation solution did not inhibit the for- This study was performed to investigate the feasibility of using mation of CaCO3 crystals despite repeated addition of the treat- low-cost commercially procurable technical-grade reagents for ment solution. Addition of media to the cementation solution large-scale MICP applications. The technical reagents served as stimulates the growth of bacterial cells during biocement treat- alternatives to analytical-grade reagents typically used in ment due to the richness of free amino acid, protein vitamins, car- most small-scale MICP experiments. The performance of bohydrates and minerals [53]. S. pasteurii in technical-grade reagents used for cultivation and The reduction of soil permeability or porosity depends on the sand biocementation was compared those of analytical-grade extent of CaCO3 crystal formation and the distribution during bio- reagents. The following conclusions can be drawn: cement treatment [29]. The biocemented samples were analysed to determine if there was any correlation between CaCO3 contents S. pasteurii showed similar performance when cultivated in and compressive strength. Generally, MICP is a biogeochemical GM-1 and GM-2, indicating that technical-grade cultivation process that utilizes microbiological methods to mediate the media can serve as a replacement to standard laboratory improvement of geological materials used in engineering [24]. cultivation medium typically used to grow a wide range of The findings of this study showed that the use of surface percola- microorganisms. tion treatment of soil samples through an ureolysis-driven MICP The efficacy of using technical-grade cementation solutions process was able to enhance the CaCO3 content. The biocemented (0.25–1.0 M) to improve soil strength showed comparable out- samples (Supplementary materials, Fig. OA4) were analysed to comes, with respect to surface strength and CaCO3 content, with determine if there was any correlation between CaCO3 content those treated with analytical-grade solutions. (Fig. 9) and surface strength. Overall, a correlation between the Cost analysis indicated that soil biocementation through surface surface strength and CaCO3 content of the samples was observed percolation treatment favoured the use of technical-grade with the use of difference concentrations of cementation solutions. cementation reagents (0.07–0.26 US$/L) over analytical-grade

The results show improvement in surface strength and CaCO3 reagents (3.33–13.29 US$/L) with a cost difference of 47–51 content as the concentration of cementation solutions increased. fold. A.I. Omoregie et al. / Construction and Building Materials 228 (2019) 116828 15

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Chu, Applications of microorganisms to geotechnical engineering This work was funded by the School of Research Office, Swin- for bioclogging and biocementation of soil in situ, Rev. Environ. Sci. Biotechnol. burne University of Technology Sarawak Campus under Swinburne 7 (2008) 139–153, https://doi.org/10.1007/s11157-007-9126-3. Sarawak Research Grants (SSRG 2-5162, SSRG 2-5535 and SSRG 2- [21] J.T. DeJong, K. Soga, S.A. Banwart, W.R. Whalley, T.R. Ginn, D.C. Nelson, B.M. Mortensen, B.C. Martinez, T. Barkouki, Soil engineering in vivo: harnessing 5301). The authors are thankful to Nurnajwani Senian (PhD natural biogeochemical systems for sustainable, multi-functional engineering Candidate-Civil Engineering) for her assistance to procure the solutions, J. R. Soc. Interface. 8 (2011) 1–15, https://doi.org/10.1098/ technical-grade reagents used in this study. rsif.2010.0270. [22] L. Cheng, M.A. Shahin, R. 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(A) (B)

Fig. OA1: Different cultivation medium used to grow ureolytic bacterial strain. (A) Growth Medium-1 contained low-cost ingredients serving as an alternative nutrient medium while (B) Growth Medium-2 contained more expensive microbiological-grade ingredients typically used for cultivation of microorganisms.

Fig. OA2: CaCO3 precipitates after treatment with technical-grade (left) and analytical- grade (right) cementation solutions.

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Fig. OA3: Samples after treatment with S. pasteurii cultures and 1 M of cementation solutions by surface percolation.

Fig. OA4: Biocemented samples treated with ureolytic bacterial cultures and cementation solutions.

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Fig. OA5: SEM analysis of the untreated sand sample.

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Table OA1: Physicochemical compositions of the technical-grade cementation reagents Reagents Brand Properties Application name Yeast extract FD00-A yeast extract (50%), maltodextrin used in food industry (previously (40%), monosodium glutamate for seasoning of meat known as (8.8%), disodium 5’-ribonucleotide products, flavouring FB00) (1.2%), moisture (3.90%), Sodium of biscuits and baking chloride (4%), pH (5.72), canary of cakes. yellow colour and very soluble in water. Urea N46 nitrogen (46%), biuret (1%), used in agriculture moisture (0.50%) purity as urea industry as nitrogen (98%), pH (7.1), granular form, pure fertilizer and white colour and very soluble in chemical industry as water. raw material for production of plastic and resins. Calcium LONGYI calcium chloride (77%), sulphate used in chemical and chloride (0.2%), sodium chloride (6%), construction industry magnesium chloride (0.5%), calcium for de-icing, dust hydroxide (0.2%), pH (8.2), flake control, concrete form, pure white colour and very acceleration and tire- soluble in water. weighting.

186

Table OA2: Initial pH of cementation solutions containing different concentration reagents Treatment Concentration of cementation reagents solution 0.25 M 0.50 M 0.75 M 1.0 M aAG (DW) 5.42 ±0.12 5.22 ±0.13 5.10 ±0.20 5.05 ±0.04 bAG (TW) 5.40 ±0.03 5.28 ±0.09 5.24 ±0.08 5.23 ±0.02 cTG (DW) 4.53 ±0.03 4.50 ±0.04 4.44 ±0.04 4.36 ±0.03 dTG (TW) 4.62 ±0.15 4.65 ±0.14 4.58 ±0.05 4.88 ±0.03 aAnalytical-grade cementation reagents in deionized water. bAnalytical-grade cementation reagents in tap water. cTechnical-grade cementation reagents in deionized water. dTechnical-grade cementation reagents in tap water.

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Chapter 5

Scale-up production of Sporosarcina pasteurii cells using custom-built stainless steel stirred tank reactor

The contexts of this chapter presented herein contain peer-reviews and editorial edits. The paper was reprinted from Biocatalysis and Agricultural Biotechnology, Omoregie, A. I., Palombo, E., Ong, D. E. L., & Nissom, P. M. (2020), ‘A feasible scale-up production of Sporosarcina pasteurii using custom-built stirred tank reactor for in-situ soil biocementation’.

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Biocatalysis and Agricultural Biotechnology 24 (2020) 101544

Contents lists available at ScienceDirect

Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

A feasible scale-up production of Sporosarcina pasteurii using custom-built stirred tank reactor for in-situ soil biocementation

Armstrong I. Omoregie a,*, Enzo A. Palombo b, Dominic E.L. Ong c, Peter M. Nissom a,d

a School of Chemical Engineering and Science, Swinburne University of Technology, Sarawak Campus, Jalan Simpang Tiga, 93350, Kuching, Sarawak, Malaysia b Department of Chemistry and Biotechnology, Swinburne University of Technology, Hawthorn, Victoria, 3122, Australia c School of Engineering and Built Environment, Griffth University, Brisbane, Queensland, 4122, Australia d Sarawak Research and Development Council, C/O Ministry of Education, Science and Technological Research, LCDA Tower, Off Jalan Bako, 93050, Kuching Sarawak, Malaysia

ARTICLE INFO ABSTRACT

Keywords: Production of urease enzyme from ureolytic bacteria (e.g. Sporosarcina pasteurii) is essential for precipitation of Non-sterile cultivation CaCO3. This bioprocess is notably utilized for soil improvement, biocementation and bioremediation of heavy Soil biocalcifcation metals. Despite the viability for feld-scale implementation as a suitable alternative to conventional treatment Crystal morphology methods, most reports in the literature are constrained to laboratory-scale. Thus, this study demonstrates an Large-scale production economic strategy to scale-up the production and cultivation of ureolytic bacteria using a custom-built stainless Urease enzyme steel stirred tank reactor (3 m3). Scalability of the bacterial cells was carried out from 214 L to 2400 L seed cultures for 90 h. Technical-grade ingredients (food-grade yeast extract media and chemicals) were used for scale-up production of the bacterial cells. The growth profle, pH and urease activity were subsequently moni tored throughout the in-situ production period. Biocementation on sand mould specimens was also performed using bacterial cultures obtained from the reactor and low-grade cementation reagents. The sand columns were grouped into different treatment cycles to determine the effect of ureolysis. CaCO3 content, effuent pH and microstructural analyses (scanning electron microscopy with energy dispersive X-ray spectroscopy) of the treated soil samples were also evaluated. The results show that scale-up production of ureolytic bacteria cells under non- sterile conditions using a custom-built reactor for industrial MICP application is feasible.

1. Introduction commercial growth medium sterilization (i.e. $0.46 to $0.66 per litre) which can be twice the cost of growth medium ingredients (Cheng and Microbially induced carbonate precipitation (MICP) relies on mi Cord-Ruwisch, 2012; Whiffn, 2004). crobial agents for nucleation sites provided by the bacterial cells to Most laboratory-based experiments on scalability of ureolytic bac ensure suffcient urease production and CaCO3 precipitation. Several terial are performed in chemostats (1 L–5 L) (Zhu and Dittrich, 2016). studies have shown that Sporosarcina pasteurii is a preferred ureolytic Unfortunately, for feld-scale trials, larger reactors will be required bacterium for numerous MICP studies because it can tolerate harsh which can be cost-prohibitive. Lately, some researchers have recom environmental conditions and still produce desirable urease (Gowtha mended the use of custom-built reactors and non-sterile bacterial man et al., 2019; Singh et al., 2017; Stocks-Fischer et al., 1999; Whiffn cultivation methods which may help minimize the production cost by et al., 2007). Despite the successful potential of MICP technology, only more than 50% (Cheng and Cord-Ruwisch, 2013; Wang et al., 2017; Zhu few feld-scale trials are presently available in the literature (Esnault- and Dittrich, 2016). However, only few studies have demonstrated po Filet et al., 2012; Gomez et al., 2019, 2015; Phillips et al., 2018; van tential strategies that can be adopted to produce large volume of ure Paassen, 2009). The cost of cultivation materials and sterility require olytic bacteria for practical implementation. Whiffn (2004) reported ment for ureolytic bacterial production adversely affect the imple using a custom-built fbreglass airlift pilot-scale reactor (120 L capacity) � mentation of MICP. Previous studies have also reported that the cost to to scale-up the cells of S. pasteurii for 50 h at 30 C under non-sterile scale-up bacterial cells can reach 60% of the total operating costs and conditions. Cheng and Cord-Ruwisch (2013) showed that batch-scale

* Corresponding author. E-mail address: [email protected] (A.I. Omoregie).

https://doi.org/10.1016/j.bcab.2020.101544 Received 17 November 2019; Received in revised form 7 February 2020; Accepted 13 February 2020 Available online 22 February 2020 1878-8181/© 2020 Elsevier Ltd. All rights reserved. A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101544

chemostats (1 L capacity) could stimulate bacterial growth from acti � vated sludge samples under non-sterile conditions at 28 C for 140 h. Recently, Aoki et al. (2018) reported using a low-technology down-fow hanging sponge reactor (170 cm3 capacity) to enrich ureolytic micro � organism from a reservoir-collected sample at 25 C for 130 days under non-sterile conditions. However, MICP researchers are yet to report on in-situ cultivation of ureolytic bacterial cells using a custom-built stainless steel stirred tank reactor (3 m3). The aim of the study herein was to investigate the feasibility of scaling up the production of S. pasteurii strain under non-sterile condi tions for in-situ soil biocementation. The bacterial cells were scaled -up from 214 L to 720 L and fnally 2400 L for 90 h. Next, in-situ bio cementation on sand mould samples via a simple surface percolation method and different treatment cycles was performed to determine the performance of the bacterial cells after cultivation. CaCO3 content, effuent pH and scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDX) analyses of the treated soil speci mens were subsquently evaluated after soil biocementation.

2. Materials and methods

2.1. Ureolytic bacterial strain

S. pasteurii NB28(SUTS) (GenBank accession number KX212192.1), previously isolated by Omoregie et al. (2017) was used throughout this study. Freeze-dried culture stock was rehydrated in sterile nutrient broth

(13 g L 1, HiMedia, Laboratories Pvt. Ltd). The revived bacterial cells

were then grown on Tryptone Soya Agar (TSA; 40 g L 1, HiMedia, 3 Laboratories Pvt. Ltd) and urea (20 g.L 1, Merck, Shd. Bhd) overnight at Fig. 1. Digital image of the custom-built stirred tank reactor (3 m ) used for � large-scale production of ureolytic bacteria. 32 C.

2.2. Inoculum culture preparation Table 1 Geometric parameters of the custom-built stirred tank reactor. The production of microbial inoculum for bulk starter culture was Parameter Result adopted from Prabakaran and Balaraman (2006) with minor modifca tions. Initially, bacterial colonies from the TSA slant was inoculated into Brand/Catalog of tank King Kong/HR300 Shape of tank Vertical round bottom cultivation medium that contained technical-grade yeast extract (15 g. 1 1 Typical usage of tank Water storage L , Angel Yeast Co. Ltd.) ammonium sulphate (10 g.L , Petronas Total capacity 3000 L 1 Chemical Company Bhd), sodium acetate (4.1 g.L , Petronas Chemical Working capacity 2400 L

Company Bhd.), urea (40 g.L 1, Petronas Chemical Company BhD.) and Diameter of tank 1470 mm pH 7.0 (Omoregie et al., 2019a). The cultures were then incubated for Height of tank 3230 mm � Ratio height of liquid to height of reactor (HL/Ht) 0.8 48 h at 32 C with shaking (150 rpm) on an orbital shaker (Smith, Ratio height of reactor to diameter of tank (HL/Dt) 2.2 A3446). Next, freshly prepared sterile cultivation medium (1.5 L) were Ratio diameter of impeller to diameter of tank (Da/Dt) 0.5 placed inside 8 separate Erlenmeyer fasks (3 L capacity), inoculated Thickness of tank 2 mm � with the previously grown bacterial cultures and incubated at 30 C for Diameter of tank cover 40 mm Diameter of outlet 25 mm 24 h under shaking condition (110 rpm) (CERTOMAT® CT plus, Agitation speed 35 rpm Sartorius). Similar to Gomez et al. (2016) and Phillips et al. (2018), the Agitator power 0.75 kW bacterial cultures (65 L) were cultivated daily and transferred for stor Quantity of aeration pods 4 age. Three separate vented anti-glug plastic water containers (30 L ca Quantity of impeller 2 pacity) were then selected and disinfected to temporarily house the Material of impeller Stainless steel Diameter of impeller 800 mm accumulated bacterial cultures. After 120 h, 13 L of the bacterial cul Height of impeller shaft 2286 mm tures were transferred into the plastic containers and stored in a cold Thickness of elastomeric nitrile rubber insulation 6 mm � room (4 C) before being used for in-situ scalability process. The bulk ureolytic bacterial cultures had optical densities (OD) ranging from 0.7 to 1.2 and pH ranging from 9.2 to 9.6. clean tap water to remove chloride residues (Seifan et al., 2017). From the total reactor volume, 80% was selected for bacterial cultivation to 2.3. Setup of the custom-built reactor minimize possible build-up of gas bubbles which may occur during fermentation (Couper et al., 2009; Gossain and Mirro, 2010). The strategy to scale-up the cells of S. pasteurii was conducted under non-sterile condition in a custom-built stirred tank reactor (3 m3) (Fig. 1 2.4. Large-scale ureolytic bacterial cultivation and Table 1) and the vessel was fabricated by Creative Encounter Sdn Bhd, Malaysia. Total production cost for the main components used to For step-wise bacterial production from 214 L, 720 L and 2400 L seed fabricate the stirred tank reactor is USD 6574.97 (Supplementary ma cultures, approximately 30% (v/v) inoculate was used. The growth terials, Table AO1). Prior to cultivation, the vessel interior section was medium ingredients used in this study were of technical-grade and chemically sterilized with liquid detergent and sodium hypochlorite prepared in a disinfected plastic container (200 L capacity) with clean solution (5%, v/v) (Whiffn, 2004). The vessel was later rinsed with tap water. The physicochemical properties of the tap water

2 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101544

(Supplementary materials, Table AO2) was analysed by EnviC Labora Table 2 tory Sdn. Bhd. Malaysia. The initial seed culture (216 L) was prepared Experimental conditions for the sand mould sets. 1 1 with yeast extract (30 g.L ), urea (40 g.L ), sodium acetate (4.1 g. Mould Sand Cementation Bacterial culture Treatment 1 1 L ), ammonium sulphate (10 g.L ) and transferred into the reactor via sets solution duration 1 a liquid transfer pump (Pedrollo; PQ60, 40 L min ). The bacterial cells 1 5 kg 3 L (once per day) 2.5 L (twice only on 3 days were acclimatised for 30 h before subsequently increasing the volume to frst day)

720 L (20 g.L 1 of yeast extract, 10 g.L 1 of urea and 10 g.L 1 of 2 5 kg 3 L (once per day) 2.5 L (twice only on 6 days

ammonium sulphate) and 2400 L (20 g.L 1 of yeast extract and 10 g.L 1 frst day) 3 15 9 L (once per day) 7.5 L (twice only on 9 days of ammonium sulphate). Both seed cultures were also incubated for 30 h kg frst day) with shaking condition (35 rpm). The cost analysis for the ingredients used for the mass production of the ureolytic bacteria is shown in Sup plementary materials, Table AO3. dissolving in 200 mL of 1.5 M HCl with continuous stirring to completely dissolve the CaCO3 (Mahawish et al., 2019). The specimens were then 2.5. Monitoring methods washed with sterile deionized water for 10 min through a Whatman® � flter paper (11 μm pore size) and oven-dried at 90 C for 24 h (Osinubi Performance of the ureolytic bacterial cells was monitored et al., 2019). CaCO3 content was determined by weight difference before throughout the fermentation period. Samples (40 mL) were taken every and after being subjected to acid wash. For each test, nine treated soil 6 h from the sampling collection outlet, placed in sterile 50 mL tubes and specimens were measured to achieve average values. transported on ice to the laboratory. Analytical assay methods previ ously described by Omoregie et al. (2019a) were used to determine the 2.8. Microstructure characterization growth profle (OD at 600 nm using a Thermo Scientifc™ GENESYS™ 20 spectrophotometer), pH profle (SevenEasy™ (Mettler Toledo pH For investigation of the MICP performance in the sand moulds after meter with precision of 0.01) and urease activity (electric conductivity biocementation treatment, samples were imaged using SEM-EDX

meter (Milwaukee, MI806, with a precision of 1 μS cm 1) of the culture (Hitachi S–3400N) analyses. Samples were sputter-coated with a thin samples. Dissolved oxygen (DO), total dissolved solids (TDS) and salinity layer of gold prior to analysis. The SEM analysis was performed at an of bacterial cultures were also monitored during the cultivation period. accelerating voltage 15 kV, a spot size of 3.0, magnifcation 5x up to The DO, TDS and salinity were measured with disinfected multipurpose 300,000x and resolution of 3.0–4.0 nm, respectively. EDX spectroscopy meter (YSI Pro Plus multiparameter meter, Xylem) having a precisions of was used to determine the elemental composition of samples.

0.05%, 0 mg.L 1 and 0.1 ppt, respectively. In addition, the interior � temperature (23–34 C) and relative humidity (60–100%) of the 2.9. Statistical analysis custom-built reactor were observed throughout the cultivation period (Supplementary materials, Table AO4). The experimental results were analysed using the statistical analysis software GraphPad Prism® (version 8) to determine the signifcance of 2.6. Biocementation by surface percolation differences between the means groups obtained in this present study. One-way analysis of variance (ANOVA) and Tukey-Kramer post hoc test The sandy soil (pH 7.16 � 0.02) used herein was sourced from Phua were performed using Minitab® (version 19). The level of signifcance Kheng Heng while polystyrene boxes served as moulds for the bio was set at 0.05. cementation experiment. The treatment had different cycles for each mould group. Mould 1 (24.38 cm height, 34.04 cm length and 25.15 cm 3. Results width) mould 2 (13.97 cm height, 22.10 cm length and 22.10 cm width) and mould 3 (21.59 cm height, 22.86 cm length and 16.51 cm width) 3.1. Scale-up cultivation of ureolytic bacteria were each flled with 15 kg, 5 kg and 5 kg of sand, respectively. Ac cording to Unifed Soil Classifcation System, the sand used in this study Prior to the in-situ bacterial cultivation, the performance of was classifed as poor-grade soil (Supplementary materials, Table AO5). S. pasteurii NB28(SUTS) in sterile (OD of 1.1 � 0.02 and urease activity

Four silicone tube hoses (30 cm length) and scouring pads (porous of 19.2 � 0.04 mM urea hydrolysed min 1) and non-sterile (OD of 0.9 �

plastic pads) were attached to all moulds before discharging the effuent. 0.07 and urease activity of 14.5 � 0.02 mM urea hydrolysed min 1) The cementation solution (CS, pH 5.01 � 0.03) used for MICP treatment cultivation medium using technical-grade ingredients in tap water was

via surface percolation consisted of technical-grade urea (66.06 g.L 1), investigated under laboratory-conditions (data not shown). Cultivation

calcium chloride (110 g.L 1, Lianyungang Longyi Industry Co. Ltd) and in non-sterile medium resulted in 18% and 25% reduction for OD and

yeast extract (5 g.L 1). The moulds were frst percolated with bacterial urease activity, respectively, when compared to sterile cultivation me cultures and allowed to settle for 2 h before adding the technical-grade dium. Hence, the 65 L inoculum was prepared under sterile laboratory- CS. The bacterial cultures were added into the mould sets twice only on condition to minimize contamination. Fig. 2 shows the scale-up process the frst day while the CS were added once per day. Repeated treatment of the ureolytic bacterial cultures. cycles (8 days, 5 days and 2 days for mould 1, mould 2 and mould 3, The results obtained for biomass, pH and urease activity for the respectively) were carried out with only CS until the percolation period ureolytic bacteria cells are presented in Fig. 3 and Table 3. The was complete (Table 2). Effuent samples (50 mL) were periodically maximum OD for 216 L, 720 and 2400 L seed cultures were 0.5 � 0.00, taken from the ejection points for pH analysis while the rest were 1.6 � 0.02 and 1.8 � 0.09, respectively. The maximum pH and urease properly discarded. Each mould was then fushed with clean tap water to activity values for 216 L seed culture were 0.4 � 0.05 and 9.3 � 0.14 mM

remove any soluble ammonium salt formed after which they were urea hydrolysed min 1, while those of 720 L seed culture were 9.1 �

allowed to cure for 8 weeks before further analyses. 0.02 and 1.1 � 0.13 mM urea hydrolysed min 1, then fnally those of 2400 L seed culture were 9.2 � 0.05 and 11.1 � 0.48 mM urea hydro 1 2.7. Measurement of CaCO3 content lysed min , respectively. Measurements of DO, TDS and salinity for samples collected from the reactor during incubation are shown in Acid washing was used to estimate the amount of CaCO3 deposited in Table 4. Also, culture samples collected from the reactor were screened the sand mould (Omoregie et al., 2019c). Specimens (100 g) were taken for the presence of bacterial contaminants. Phenotypic and molecular within 20–30 mm from the top, middle and bottom sections before characterizations were carried out following methods described by

3 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101544

Table 3 Growth kinetics of ureolytic bacteria after cultivation. Kinetics parameters Microbial seed cultures

216 L 720 L 2400 L

Specifc growth rate (h 1) 0.5 � 0.00 0.0 � 0.00 0.0 � 0.00 Doubling time (h) 14.4 � 0.7 55.1.�2.4 23.6.1.�2.5 Maximum OD600 0.5 � 0.00 1.6 � 0.02 1.8 � 0.09 Maximum pH 9.4 � 0.05 9.1 � 0.02 9.2 � 0.05

Urease activity (mM urea hydrolysed.min 1) 9.3 � 0.14 11.1 � 0.13 1 11.1 � 0.48

Omoregie et al. (2016) and Omoregie et al. (2019b). Four ureolytic 3.3. Microstructure investigation bacterial isolates with accession numbers MK595743, MK595744 MK595745 and MK595747 were identifed as Pseudogracilibacillus SEM with EDX analyses were performed on the treated samples marinus. collected from the top layer of the crushed biocemented sand specimens. The SEM micrographs showed that the treated specimens were coated with CaCO3 mesocrystals which, in some cases, bridged the sand parti 3.2. Soil biocementation and CaCO3 content cles together (supplementary materials, Fig. AO3 and Fig. AO4). The SEM micrographs (Fig. 5A–C) also showed that different treatment pe After MICP treatment, white precipitates of crystals manifested on riods infuenced the crystals morphological formations. In Fig. 5A, the surfaces of some sand mould specimens as a result of ureolysis (Supplementary materials, Fig. AO1). This outcome meant that the bacterial cells grown under non-sterile conditions could produce suff cient urease to induce cementitious material for future large-scale MICP trials (Supplementary materials, Fig. AO2). Table 5 shows pH analysis of effuent samples that were spatially collected during soil biocementation to monitor ureolysis activity as the treatment period progressed. In summary, the pH from mould 1 samples decreased from 8.7 � 0.09 to 8.0 � 0.05 after 3 days, while pH from mould 2 samples decreased from 8.7 � 0.18 to 7.4 � 0.10 after 6 days. The result also showed that pH from mould 3 samples decreased from 8.7 � 0.11 to 7.2 � 0.06. The distribution of CaCO3 at top, middle and bottom layers of the treated sand specimens are shown in Fig. 4. Total CaCO3 contents increased progressively with treatment cycles which was applied on different mould sets. The difference of CaCO3 content between the top and bottom layers of mould 1 (3.8%) was lower than those of mould 2 (7.2%) and mould 3 (7.0%). The distribution of CaCO3 in mould 1 was 9.2 � 0.54% (top-layer), 4.5 � 0.44% (middle-layer) and 5.4 � 0.60% (bottom-layer). For mould 2, the CaCO3 distribution was 14.4 � 0.51% (top-layer), 7.2 � 0.64% (middle-layer) and 7.2 � 0.44% (bottom- layer). Finally, that of mould 3 was 18.2 � 0.70% (top-layer), 10.0 � 0.50% (middle-layer) and 11.2 � 0.68% (bottom-layer). In addition, Fig. 4 shows that moulds 1, 2 and 3 had CaCO3 content of 19.0 � 1.58%, 28.8 � 1.59% and 39.4 � 1.88%, respectively. Statistical analysis using one-way ANOVA showed that there were signifcant differences (P-value is 0.00) among the group means of the results.

Fig. 2. An inner view of the custom-built reactor during scale-up process of the Fig. 3. Growth profle (A); pH profle (B) and urease activity (C) analyses for bacterial cells. Sequentially, volume of cultures was increased from 216 L (A) to ureolytic bacterial cultivation. The vertical error bars represent mean � SD (n 2400 L (B). ¼ 9).

4 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101544

Table 4 DO, TDS and salinity of the bacterial seed culture samples.

Time (h) 216 L seed culture 720 L seed culture 2400 L seed culture

DO (%) TDS (mg.L 1) Salinity (ppt) DO (%) TDS (mg.L 1) Salinity (ppt) DO (%) TDS (mg.L 1) Salinity (ppt)

0 11.2 � 1.13 4830.0 � 7.91 8.5 � 0.07 7.5 � 0.07 5639.9 � 6.15 11.7 � 0.13 6.4 � 0.05 9813.4 � 4.76 12.5 � 0.05 6 7.7 � 0.08 5092.6 � 7.67 11.9 � 0.04 5.5 � 0.07 7840.2 � 5.45 14.5 � 0.12 4.3 � 0.11 11425.0 � 7.21 17.4 � 2.12 12 6.3 � 0.03 9535.0 � 6.45 15.3 � 0.09 5.1 � 0.05 9457.4 � 4.51 16.8 � 0.14 3.3 � 0.04 17114.3 � 2.52 22.8 � 0.35 18 6.1 � 0.02 9869.3 � 14.36 18.3 � 0.10 5.4 � 0.05 11387.0 � 18.95 22.9 � 0.47 3.0 � 0.12 21005.0 � 3.46 35.2 � 0.19 24 5.9 � 0.15 11019.8 � 14.59 20.6 � 0.09 4.9 � 0.11 15683.4 � 8.78 23.8 � 0.42 3.1 � 0.09 23112.7 � 3.06 44.3 � 1.08 30 5.4 � 0.05 13937.7 � 10.25 27.4 � 0.14 4.6 � 0.10 18686.8 � 25.65 24.6 � 0.14 3.1 � 0.04 23955.0 � 3.46 51.2 � 0.86

Table 5 4. Discussion pH analysis of effuents samples collected from the mould sets.

Treatment day Mould 1 Mould 2 Mould 3 4.1. Scale-up of ureolytic bacteria in the custom-built reactor

1 pH 8.7 � 0.09 pH 8.7 � 0.18 pH 8.7 � 0.11 2 pH 8.5 � 0.07 pH 8.5 � 0.19 pH 8.5 � 0.10 Non-sterile growth of microorganisms has been widely used for 3 pH 8.0 � 0.05 pH 8.0 � 0.13 pH 8.1 � 0.09 various bioprocess such as biogas production and wastewater treatment 4 ND pH 7.8 � 0.10 pH 7.7 � 0.10 but the use of non-sterile conditions to cultivate bacteria for enzyme � � 5 ND pH 7.5 0.10 pH 7.5 0.06 production (i.e. urease) has not been widely explored (Gorski, 2012). It 6 ND pH 7.4 � 0.10 pH 7.5 � 0.07 7 ND ND pH 7.5 � 0.12 8 ND ND pH 7.4 � 0.06 9 ND ND pH 7.2 � 0.06

ND ¼ not determined.

Fig. 4. CaCO3 content of different layers (top, middle and bottom) for the biocemented sand samples. One-way ANOVA with Tukey-Kramer post hoc analyses were used to determine statistical signifcant (P < 0.05). The rectan gular bars that share same letter indicates there are no statistical signifcant differences between their means. The vertical error bars represent mean � SD (n ¼ 9).

crystals ranging from 15 μm to 25 μm with topography showing an elongated fower-like crystals having smooth appearances clustered together were seen on mould 1 specimen. In Fig. 5B, several regular crystals ranging from 15 μm to 35 μm with topography showing a cubic- like crystals having smooth appearances which are clustered together were seen on mould 2 specimen. However, Fig. 5C shows several irregular crystals structures ranging from 2.5 μm to 30 μm with topog raphy showing small cubic-like crystals scattered on the surfaces of specimen 3. Additionally, a few larger projecting cubic-like crystals clustered together with rough appearance were also seen. More so, higher SEM magnifcation (supplementary materials, Fig. AO5) showed morphological shape with elongated rhombohedral, elemental rhom Fig. 5. SEM micrographs showing different morphological crystal formations bohedral and polyhedral CaCO3 crystal structures for moulds 1, 2 and 3, for Mould 1 (A); Mould 2 (B) and Mould (C) after successful MICP treatment (3, respectively. 6 and 9 days respectively).

5 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101544 is worth noting that this is the frst study reporting the use of a environmental conditions akin to the feld-scale MICP trials which were custom-built reactor (3 m3) for scalability of ureolytic bacteria cells. The carried out by other researchers (Esnault-Filet et al., 2012; Gomez et al., reactor was specifcally built for simple, economic, aerobic microbial 2016, 2015; Phillips et al., 2018; van Paassen et al., 2010; Van Paassen production scale-upprocess and was situated outdoor to mimic et al., 2009). Small-scale moulds were used in the present study which is feld-scale conditions. The custom-built reactor was equipped with a contrary to the large-scale moulds (i.e. 1.7 m3 diameter tanks and 100 top-driven agitator system (stirrer motor) supported with a stand, stirrer m3 sandbox) reported in the literature. The small-scale soil biocement shaft, impellers, sampling port, air delivery system and dump port for test was to done to determine the performance of the bacterial cultures the discharge of culture. after cultivation in the custom-built reactor. Also, the use of surface The growth profle showed that there was a long lag phase which percolation method for MICP treatment is widely known and has been occurred only in the 216 L seed culture. The lag phase is the delay before reported suitable for columns below 2 m height and one-dimensional the start of exponential growth and represents the earliest and most vertical cementation treatment because they are often confned by the poorly understood stage of microbial growth cycle due to limited studies column walls (Cheng and Cord-Ruwisch, 2014, 2012). Therefore, in-situ which describe the fundamental physiological and molecular processes surface percolation was utilized since the heights of the moulds in this (Rolfe et al., 2012). When cells in stationary phases are introduced into a present study ranged from 0.14 m to 0.24 m. new enriched-nutrient environment, it is desirable for the cells to rapidly The effuent pH was monitored during treatment to monitor ure obtain biomass to secure resources before their competitors (Schultz and olysis MICP treatment which is an effective indicator for urease activity Kishony, 2013). It is not known why the 216 L seed culture displayed an changes (Gomez et al., 2016; Porter et al., 2017; Stocks-Fischer et al., extended lag phase and took a considerably longer period to reach 1999). In our study, the effuent pH ranged from 7.2 � 0.06 to 8.7 � 0.11 exponential phase when compared to 720 L and 2400 L seed cultures. It which is similar to the results reported by Porter et al. (2017) and Gomez is likely that S. pasteurii required a longer period to acclimatize to its new et al. (2016). It was suggested that the loss of urease activity may be environment and compete for nutrients with other microbial cells. attributed to biological and chemical reactions (van Paassen, 2009). During the scale-up process, urea and ammonium sulphate were Therefore in situations where urease activities are low, more urease omitted in the fnal stage of the microbial seed cultivation to reduce activity can be supplied to the soil to improve biocementation by build-up of ammonia (due to ureolysis) and decreased alkalinity during percolating more bacterial cells (Cheng et al., 2017). However, the the fermentation period. Unfortunately, one of the major problems disadvantage of such an approach is formation of unwanted clogging at affecting MICP is the production ammonia during treatment of bacterial the surfaces of the soil mould that can inhibit further penetration of cultivation which can have adverse impacts on human health and the CaCO3 precipitates during treatment. environment (Ivanov et al., 2019). It was previously determined that 62 3 kg of calcium carbonate per 1 m from soil biocementation can result in 4.3. CaCO3 content the production of 10.5 kg and 11.2 kg of ammonia into the air, groundwater or surface water (Ivanov and Stabnikov, 2017). If a sig After soil biocementation, it is common to investigate the distribu nifcant amount of urea was used during the large-scale bacterial pro tion of CaCO3 content present within the treated sand body. The top, duction (0.27 m3) in the reactor, it would release up to 0.3 km3 of middle and bottom layers of the mould specimens did not show homo ammonia gas into the atmosphere. Besides, the custom-built reactor had geneous distribution of CaCO3 precipitates. This can often be linked to no integrated systems for absorbing or treating the released gas, hence bioclogging formation during MICP treatments especially at the entry low urea concentration was used for 217 L and 720 L seed cultivation to point of injection. The available urease from the bacterial cells situated minimize ammonia production. Interestingly, Aoki et al. (2018) recently at the injection points hydrolyses urea to form CaCO3 precipitates which showed the ammonia gas could be treated during cultivation of ureolytic occurs continuously and results in a bioflm layer, thus preventing bacteria in a non-sterile bioreactor system by transferring the ammonia further cementation solutions from penetrating the soil particles. Such gas from an effuent reservoir tank (after collection from the reactor) bioclogging can prevent suffcient cementation from reaching the depth and then into a sulphuric acid solution tank. of the mould specimens (van Paassen, 2009; Whiffn, 2004). Cheng and After fnal cultivation (2400 L), the seed culture had an OD of 1.8 � Cord-Ruwisch (2014) suggested bioclogging formation during surface 0.09 which is within the recommended cell density (OD 0.8 to 2.0.) for percolation treatment is often due to immobilization of high concen MICP experiments (Al Qabany et al., 2012; Gat et al., 2014; Kashizadeh trations of bacterial cells at the injection end. Several factors such as et al., 2020). The urease activity (11.1 � 0.48 mM urea hydrolysed environmental temperature, relative humidity and reactions with native 1 min ) and pH (9.2 � 0.05) measurements showed that bacterial cells microorganisms during treatments can also infuence the rate of CaCO3 were still able to produce urease needed for CaCO3 precipitation. The precipitation. The CaCO3 content from the sample groups showed that results also indicated that under non-sterile cultivation conditions and signifcantly more crystal formation occurred in the soil moulds treated without increasing the initial pH of the medium or using high a urea for 9 days than those treated for 3 and 6 days. It is important to give concentration, ureolytic bacterial cells can be produced in large volume. cementation cycle suffcient treatment interval to allow nucleation site The use of this custom-built reactor and technical-grade reagents for and crystal formation essential for soil particle bridging to occur. large-scale bacterial production under non-sterile operation can serve as Percolated soil columns effectively lead to more CaCO3 or compressive a suitable concept for future practical experiments that have fnancial strength when a lower degree of cementation treatment cycles and constraints and are not concerned with the possibility of contaminating saturation zones are performed (Cheng et al., 2017; Cheng and microbial species. Cord-Ruwisch, 2013).

4.2. MICP treatment 4.4. Microstructural analysis

The entire soil mould specimens achieved cementation and were Microstructural analysis was undertaken to show the infuence of subjected to further analyses after curing (8 weeks). For successful different MICP treatment cycles on the sand mould specimens. SEM biocementation, it is important to introduce bacterial cells into the soil images revealed that the crystal deposits occurred on sand grains and at for proper immobilization before applying the cementation solution regions which cemented sand grains together. This happens because (Cheng and Cord-Ruwisch, 2012). The bacterial cells were able to bacterial cells can readily attach themselves to the corners of sandy soil hydrolyse urea which later allows precipitation of CaCO3 upon the and not be easily disintegrated (Porter et al., 2018). The observed SEM presence of a calcium source. The soil biocementation experiment un images which displayed crystal formation on the soil particles are dertaken in this study was performed outdoors to mimic in-situ similar to other MICP studies (Cheng et al., 2017; Porter et al., 2018,

6 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101544

2017; Seifan et al., 2017). EDX results revealed peaks of calcium, oxygen and SSRG 2-5535) and Soletanche Bachy (Rueil-Malmaison, France). and carbon elements which are indicative of CaCO3 precipitation (Porter et al., 2018). The microstructural analysis of the treated biocement sand Appendix A. Supplementary data specimens suggested that MICP treatment performed under non-sterile conditions could still precipitate a suffcient amount of CaCO3 crystals. Supplementary data to this article can be found online at https://doi. However, it is noteworthy that the CaCO3 polymorphism and mor org/10.1016/j.bcab.2020.101544. phologies can be greatly infuenced by bacterial metabolic activities, cementation reagents, extracellular polymeric substance and abiotic References factors (i.e. pH, water, sunlight, oxygen, soil, humidity and temperature) (Seifan et al., 2017). Al Qabany, A., Soga, K., Santamarina, C., 2012. Factors affecting effciency of microbially induced calcite precipitation. J. Geotech. Geoenviron. Eng. 138, 992–1001. https:// doi.org/10.1061/(ASCE)GT.1943-5606.0000666. 4.5. Cost implications Aoki, M., Noma, T., Yonemitsu, H., Araki, N., Yamaguchi, T., Hayashi, K., 2018. A low- tech bioreactor system for the enrichment and production of ureolytic microbes. Pol. J. Microbiol. 67, 59–65. https://doi.org/10.5604/01.3001.0011.6144. Using a custom-built reactor in this study can offer enormous eco Cheng, L., Cord-Ruwisch, R., 2014. Upscaling effects of soil improvement by microbially nomic advantage for in-situ MICP applications. Typically, large-scale induced calcite precipitation by surface percolation. Geomicrobiol. J. 31, 396–406. production of ureolytic bacteria is essential for augmentation or injec https://doi.org/10.1080/01490451.2013.836579. Cheng, L., Cord-Ruwisch, R., 2013. Selective enrichment and production of highly urease tion of bacterial culture. The typical developmental phases associated active bacteria by non-sterile (open) chemostat culture. J. Ind. Microbiol. with scale-up microbial cultivation (Petri dish, shake fask, bench-scale, Biotechnol. 40, 1095–1104. https://doi.org/10.1007/s10295-013-1310-6. pilot-scale and large-scale) are costly (Li et al., 2015). For example, in Cheng, L., Cord-Ruwisch, R., 2012. In situ soil cementation with ureolytic bacteria by – Malaysia, the fermentation platforms for large-scale production of bac surface percolation. Ecol. Eng. 42, 64 72. https://doi.org/10.1016/j. ecoleng.2012.01.013. terial cells (i.e. 3 L, 30 L 300 L and 3000 L total capacities) cost USD 550, Cheng, L., Shahin, M.A., Cord-Ruwisch, R., 2017. Surface percolation for soil 000 to 1.3 million (personal communication with Nursyahira Binti improvement by biocementation utilizing in situ enriched indigenous aerobic and – Mohd Ghazali from Fermetec Resources Sdn. Bhd. and Julian Lim from anaerobic ureolytic soil microorganisms. Geomicrobiol. J. 34, 546 556. https://doi. org/10.1080/01490451.2016.1232766. Medigene Sdn. Bhd.). Hence, a simple low-technology reactor (USD Couper, R.J., Penney, W.R., Fair, R.J., Walas, M.S., 2009. Chemical Process Equipment: 6574.97) which could feasibly scale-up bacterial cells with minimal Selection and Design, second ed. Gulf Professional Publishing, pp. 655–675. https:// contamination was utilized in this study. Future improvement to reduce doi.org/10.1016/C2011-0-08248-0. Esnault-Filet, A., Gadret, J.-P., Loygue, M., Borel, S., 2012. Biocalcis and its applications microbial contamination and increase biomass yield may include having for the consolidation of sands. In: Grouting and Deep Mixing, pp. 1767–1780. boilers attached to interior part of the medium tank for sterilization. In https://doi.org/10.1061/9780784412350.0152. addition, low-cost commercially procurable thermocirculator with Gat, D., Tsesarsky, M., Shamir, D., Ronen, Z., 2014. Accelerated microbial-induced CaCO3 precipitation in a defned coculture of ureolytic and non-ureolytic bacteria. heating coil could be incorporated into the custom-built reactor to aid Biogeosciences 11, 2561–2569. https://doi.org/10.5194/bg-11-2561-2014. temperature control. Gomez, M.G., Anderson, C.M., Graddy, C.M.R., DeJong, J.T., Nelson, D.C., Ginn, T.R., 2016. Large-scale comparison of bioaugmentation and biostimulation approaches for biocementation of sands. J. Geotech. Geoenviron. Eng. 143, 4016124–4016136. 5. Conclusion https://doi.org/10.1061/(asce)gt.1943-5606.0001640. Gomez, M.G., Graddy, C.M.R., DeJong, J.T., Nelson, D.C., 2019. Biogeochemical changes This study investigated the feasibility of using a custom-built stirred during bio-cementation mediated by stimulated and augmented ureolytic – tank reactor (3 m3) to scale-up the production of S. pasteurii under non- microorganisms. Sci. Rep. 9, 11517 11531. https://doi.org/10.1038/s41598-019- 47973-0. sterile operation for various MICP applications. Using a sequential scale- Gomez, M.G., Martinez, B.C., DeJong, J.T., Hunt, C.E., deVlaming, L.A., Major, D.W., up strategy, the bacterial cells were successfully grown from 216 L to Dworatzek, S.M., 2015. Field-scale bio-cementation tests to improve sands. Proc. – 2400 L with suitable cell mass (OD ¼ 1.8 � 0.09), pH (9.2 � 0.05) and Inst. Civ. Eng. Ground Improv. 168, 206 216. https://doi.org/10.1680/ � 1 grim.13.00052. urease activity (11.1 0.48 mM urea hydrolysed min ). Furthermore, Gorski, L., 2012. Selective enrichment media bias the types of salmonella enterica strains in-situ soil biocementation showed that bacterial cells grown in the isolated from mixed strain cultures and complex enrichment broths. PloS One 7, 1–10. https://doi.org/10.1371/journal.pone.0034722. custom-built reactor could induce CaCO3 precipitation after being Gossain, V., Mirro, R., 2010. Linear Scale-Up of Cell Cultures: the next level in disposable immobilized in sand specimens. CaCO3 content and microstructural bioreactor design. Bioprocess. Int. 8, 56–62. analyses were able to confrm the presence of CaCO3 crystal formation Gowthaman, S., Iki, T., Nakashima, K., Ebina, K., Kawasaki, S., 2019. Feasibility study for within the treated sand particles. slope soil stabilization by microbial induced carbonate precipitation (MICP) using indigenous bacteria isolated from cold subarctic region. SN Appl. Sci. 1, 1480. https://doi.org/10.1007/s42452-019-1508-y. Declaration of competing interest Ivanov, V., Stabnikov, V., 2017. Construction Biotechnology, frst ed. Springer, Singapore. https://doi.org/10.1007/978-981-10-1445-1. Ivanov, V., Stabnikov, V., Stabnikova, O., Kawasaki, S., 2019. Environmental safety and The authors declare that they have no known competing fnancial biosafety in construction biotechnology. World J. Microbiol. Biotechnol. 35 https:// interests or personal relationships that could have appeared to infuence doi.org/10.1007/s11274-019-2598-9. the work reported in this paper. Kashizadeh, E., Mukherjee, A., Tordesillas, A., 2020. Experimental and numerical investigation on heap formation of granular soil sparsely cemented by bacterial calcifcation. Powder Technol. 360, 253–263. https://doi.org/10.1016/j. CRediT authorship contribution statement powtec.2019.09.086. Li, J., Jaitzig, J., Lu, P., Süssmuth, R.D., Neubauer, P., 2015. Scale-up bioprocess Armstrong I. Omoregie: Conceptualization, Methodology, Valida development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations. Microb. Cell Factories 14, 1–13. https://doi. tion, Formal analysis, Investigation, Visualization, Writing - original org/10.1186/s12934-015-0272-y. draft, Writing - review & editing. Enzo A. Palombo: Supervision, Mahawish, A., Bouazza, A., Gates, W.P., 2019. Unconfned compressive strength and Writing - review & editing. Dominic E.L. Ong: Funding acquisition, visualization of the microstructure of coarse sand subjected to different biocementation levels. J. Geotech. Geoenviron. Eng. 145, 4019033. https://doi.org/ Supervision, Resources, Conceptualization. Peter M. Nissom: Funding 10.1061/%28ASCE%29GT.1943-5606.0002066. acquisition, Supervision, Resources, Project administration. Omoregie, A.I., Khoshdelnezamiha, G., Senian, N., Ong, D.E.L., Nissom, P.M., 2017. Experimental optimisation of various cultural conditions on urease activity for isolated Sporosarcina pasteurii strains and evaluation of their biocement potentials. Acknowledgement Ecol. Eng. 109, 65–75. https://doi.org/10.1016/j.ecoleng.2017.09.012. Omoregie, A.I., Ngu, L.H., Ong, D.E.L., Nissom, P.M., 2019a. Low-cost cultivation of This work was fnancially supported by the School of Research Offce Sporosarcina pasteurii strain in food-grade yeast extract medium for microbially induced carbonate precipitation (MICP) application. Biocatal. Agric. Biotechnol. 17, (Swinburne University of Technology Sarawak Campus, Sarawak, 247–255. https://doi.org/10.1016/j.bcab.2018.11.030. Malaysia) under Swinburne Sarawak Research Grants (SSRG 2-5162

7 A.I. Omoregie et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101544

Omoregie, A.I., Ong, D.E.L., Nissom, P.M., 2019b. Assessing ureolytic bacteria with calcifying abilities isolated from limestone caves for biocalcifcation. Lett. Appl. – Microbiol. 68, 173 181. https://doi.org/10.1111/lam.13103. Armstrong I. Omoregie obtained his BSc (Biotechnology) and Omoregie, A.I., Palombo, E.A., Ong, D.E.L., Nissom, P.M., 2019c. Biocementation of sand MSc (Research) from Swinburne University of Technology by Sporosarcina pasteurii strain and technical-grade cementation reagents through Sarawak campus (SUTS) in 2013 and 2016, respectively. surface percolation treatment method. Construct. Build. Mater. 228, 116828. Omoregie is presently a fnal year PhD candidate at the same https://doi.org/10.1016/j.conbuildmat.2019.116828. university (SUTS) and will be submitting his thesis for external Omoregie, A.I., Senian, N., Li, P.Y., Hei, N.L., Leong, D.O.E., Ginjom, I.R.H., Nissom, P. examination in March 2020. His PhD focuses on investigating M., 2016. Ureolytic bacteria isolated from Sarawak limestone caves show high the feasibility to scale-up the production of Sporosarcina pas urease enzyme activity comparable to that of Sporosarcina pasteurii (DSM 33). teurii for cost-effective in-situ biocementation. His research – Malays. J. Microbiol. 12, 463 470. interests include biomineralization, biocementation, applied Osinubi, K.J., Eberemu, A.O., Gadzama, E.W., Ijimdiya, T.S., 2019. Plasticity biotechnology, fermentation technology and enzymology. characteristics of lateritic soil treated with Sporosarcina pasteurii in microbial- Omoregie is a member (Graduate Technologist-GT19120436) induced calcite precipitation application. SN Appl. Sci. 1, 829. https://doi.org/ of Malaysia Board of Technologist and student member 10.1007/s42452-019-0868-7. (MSM/M/15/16/SM6) of Malaysian Society for Microbiology. Phillips, A.J., Troyer, E., Hiebert, R., Kirkland, C., Gerlach, R., Cunningham, A.B., Spangler, L., Kirksey, J., Rowe, W., Esposito, R., 2018. Enhancing wellbore cement integrity with microbially induced calcite precipitation (MICP): a feld scale demonstration. J. Petrol. Sci. Eng. 171, 1141–1148. https://doi.org/10.1016/j. petrol.2018.08.012. Dominic Ek Leong Ong is a senior lecturer (School of Engi Porter, H., Dhami, N.K., Mukherjee, A., 2018. Sustainable road bases with microbial neering and Built Environment - Civil and Environmental En precipitation. Proc. Inst. Civ. Eng. Constr. Mater. 171, 95–108. https://doi.org/ gineering) at Griffth University, Australia. He obtained his 10.1680/jcoma.16.00075. BEng (Honours in Civil Engineering) from University of West Porter, H., Dhami, N.K., Mukherjee, A., 2017. Synergistic chemical and microbial ern Australia in 1998 and PhD (Engineering) from National cementation for stabilization of aggregates. Cement Concr. Compos. 83, 160–170. University of Singapore in 2004. Dr. Ong is a Fellow member of https://doi.org/10.1016/j.cemconcomp.2017.07.015. both Engineers Australia and the Institution of Engineers Prabakaran, G., Balaraman, K., 2006. Development of a cost-effective medium for the Malaysia (IEM). He is a Chartered Professional Engineer large scale production of Bacillus thuringiensis var israelensis. Biol. Contr. 36, (CPEng) and a Registered Professional Engineer, Queensland 288–292. https://doi.org/10.1016/j.biocontrol.2005.09.018. (RPEQ). He is also the co Editor-in-Chief of Geotechnical Rolfe, M.D., Rice, C.J., Lucchini, S., Pin, C., Thompson, A., Cameron, A.D.S., Alston, M., Research, a journal of the Institution of Civil Engineers (ICE), Stringer, M.F., Betts, R.P., Baranyi, J., 2012. Lag phase is a distinct growth phase that UK, as well as an Adjunct Associate Professor of Swinburne prepares bacteria for exponential growth and involves transient metal accumulation. University of Technology. J. Bacteriol. 194, 686–701. https://doi.org/10.1128/JB.06112-11. Schultz, D., Kishony, R., 2013. Optimization and control in bacterial lag phase. BMC Biol. 11, 120–123. https://doi.org/10.1186/1741-7007-11-120. Seifan, M., Samani, A.K., Berenjian, A., 2017. New insights into the role of pH and Enzo Palombo Received PhD from La Trobe University aeration in the bacterial production of calcium carbonate (CaCO3). Appl. Microbiol. studying the genetics of bacterial conjugation and undertook ’ Biotechnol. 101, 3131–3142. https://doi.org/10.1007/s00253-017-8109-8. post-doctoral training at the Royal Children s Hospital inves Singh, A.K., Singh, M., Verma, N., 2017. Extraction, purifcation, kinetic characterization tigating the genetic epidemiology of gastroenteritis viruses, and immobilization of urease from Bacillus sphaericus MTCC 5100. Biocatal. Agric. particularly rotavirus and astrovirus. Enzo, a professor of ’ Biotechnol. 12, 341–347. https://doi.org/10.1016/j.bcab.2017.10.020. microbiology, is chair of Swinburne s Department of Chemistry Stocks-Fischer, S., Galinat, J.K., Bang, S.S., 1999. Microbiological precipitation of and Biotechnology. Research interests include food microbi CaCO3. Soil Biol. Biochem. 31, 1563–1571. https://doi.org/10.1016/S0038-0717 ology, identifcation of bio-active compounds from medicinal (99)00082-6. plants, fungi and biowaste, environmental microbiology, van Paassen, L., 2009. Biogrout: Ground Improvement by Microbially Induced Carbonate nanotechnology and virology. Involved in teaching in the areas Precipitation. Doctoral Thesis. Delft University of Technology, Delft, The of microbiology, biotechnology, biochemistry and molecular Neatherlands. biology. He is Member of Dairy Industry Association of van Paassen, L.A., Ghose, R., van der Linden, T.J.M., van der Star, W.R.L., van Australia. Member of AusBiotech. Member of Environmental Loosdrecht, M.C.M., 2010. Quantifying biomediated ground improvement by Assessment and Research Consortium. ureolysis: large-scale biogrout experiment. J. Geotech. Geoenviron. Eng. 136, 1721–1728. https://doi.org/10.1061/(ASCE)GT.1943-5606.0000382. Van Paassen, L.A., Harkes, M.P., Van Zwieten, G.A., Van Der Zon, W.H., Van Der Star, W. Peter Morin Nissom obtained his B.Sc (Hons) in Microbiology R.L., Van Loosdrecht, M.C.M., 2009. Scale up of BioGrout: a biological ground from University of Malaya in 1992 and D.Phil in Biochemistry/ reinforcement method. In: Proc. 17th Int. Conf. Soil Mech. Geotech. Eng. Acad. Molecular Biology from Exeter College, University of Oxford in Pract. Geotech. Eng., 3, pp. 2328–2333. https://doi.org/10.3233/978-1-60750-031- 1998. He was a Research Scientist, and Head of Microarray 5-2328. Group with A-Star Bioprocessing Technology Institute in Wang, Z., Zhang, N., Cai, G., Jin, Y., Ding, N., Shen, D., 2017. Review of ground Singapore (2001 to 2010). Nissom is currently the General improvement using microbial induced carbonate precipitation (MICP). Mar. Manager Sarawak Research and Development Council, C/O Georesour. Geotechnol. 35, 1135–1146. https://doi.org/10.1080/ Ministry of Education, Science and Technological Research, 1064119X.2017.1297877. Sarawak Malaysia. He is also an Adjunct Associate Professor of Whiffn, V.S., 2004. Microbial CaCO3 Precipitation for the Production of Biocement. Swinburne University of Technology. His areas of expertise are Doctoral Thesis. Murdoch University, Perth, Australia. industrial and environmental microbiology, molecular and Whiffn, V.S., van Paassen, L.A., Harkes, M.P., 2007. Microbial carbonate precipitation as cellular biology, system biology and genomics with speciali a soil improvement technique. Geomicrobiol. J. 24, 417–423. https://doi.org/ zation in high throughput gene expression profling. 10.1080/01490450701436505. Zhu, T., Dittrich, M., 2016. Carbonate precipitation through microbial activities in natural environment, and their potential in biotechnology: a review. Front. Bioeng. Biotechnol. 4, 1–21. https://doi.org/10.3389/fbioe.2016.00004.

8 Supplementary materials

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Mould 2 Mould 1 Mould 1

Mould 2 Mould 3 Mould 1 Mould 2

Mould 3

Fig. AO1: Biocemented samples after cured for 8 weeks.

Fig. AO2: Overall set up of the in situ biocement treatment trial to be performed in a sand box (10 m3) using ureolytic bacterial cultured in the custom-built reactor.

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50 µm

Fig. AO3: SEM micrograph showing biocement sand grains coated with CaCO3 crystals.

199

100 µm

Fig. AO4: SEM micrograph showing the bridging of biocemented sand particles by CaCO3 precipitation.

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(A)

10 µm

(B)

10 µm

(C)

10 µm

Fig. AO5: SEM micrographs showing topographical crystal structures of the biocemented sand specimens obtained from Mould 1 (A), Mould 2 (B) and Mould (C).

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Table AO1: Cost analysis of the reactor Features Cost (USD) Custom-built reactor (Stainless steel water tank, support stand, 3377.97 painting, PVC level gauge, elastomeric nitrile rubber insulation, air pump with pods, outlet values, tubing)

Stirrer system (Stirrer motor, impeller shaft, paddle impelle) 1471.83 Control panel set-up (metal control panel, electronic inverter, 1049.58 magnetic contactor and 3 phase induction motor) Transfer pump and pipping system 675.59 Total 6574.97

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Table AO2: Physiochemical properties of tap water used in this study Parameter Result

Dissolved oxygen 6.47 mg.L-1

Biochemical oxygen demand 1.94 mg.L-1

-1 Chemical oxygen demand 39.5 mg O2.L

Total suspended solids 1.00 mg.L-1

Total dissolved solids 51.28 mg.L-1

Salinity 0.25 % pH 7.46

Chloride 5.14 mg.L-1

Fluoride 0.02 mg.L-1

Sodium 3.48 mg.L-1

Magnesium 1.11 mg.L-1

Iron 0.02 mg.L-1

Aluminium 3.89 mg.L-1

Nitrate 1.34 mg.L-1

Ammonium nitrogen 0.02 mg.L-1

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Table AO3: Cost analysis of ingredients used for cultivation of S. pasteurii Ingredients 216 L seed culture 720 L seed culture 2400 L seed culture

Concentrati *Cost Concentrati *Cost Concentrati *Cost on (kg) (USD) on (kg) (USD) on (kg) (USD)

Yeast 4.47 84.26 20.16 380.02 67.20 1266.72 extract

Urea 1.49 1.95 5.04 6.60 - -

Ammonium 1.49 5.72 5.04 19.35 16.08 61.74 sulphate

Sodium 0.61 2.56 - - - - acetate

Total cost 94.49 405.97 1328.46

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Table AO4: Temperature and humidity measurements during S. pasteurii cultivation Time Temperature (oC) Relative humidity (%) (h) 216 L 720 L 2400 L 216 L 720 L 2400 L

0 25 26 31 100 92 83

1 26 28 32 100 87 79

2 27 29 30 93 75 79

3 27 32 30 94 75 85

4 27 33 30 94 65 87

5 26 34 28 95 65 87

6 26 34 28 94 63 87

7 27 34 28 93 63 89

8 28 33 28 94 63 89

9 28 32 27 96 76 97

10 29 32 28 94 76 99

11 29 32 27 83 79 99

12 30 33 27 83 83 99

13 30 33 27 60 86 99

14 31 32 26 60 87 97

15 31 32 26 60 94 97

16 33 31 25 60 98 98

17 33 28 24 60 98 98

18 34 28 28 62 99 97

19 32 29 28 75 99 96

20 30 29 28 80 97 96

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21 29 26 32 83 96 87

22 29 25 33 99 96 67

23 29 25 33 99 99 67

24 26 25 33 95 97 65

25 25 26 32 95 96 66

26 25 26 30 95 89 68

27 25 25 33 93 87 79

28 24 24 31 95 76 85

29 24 26 28 95 77 88

30 24 26 29 96 76 86

Table AO5: Physical properties of the sand used in this study

D10 D30 D60 Cu Cc pH Specific Maximum Soil gravity dry classification

(Gs) density USCS

(ρd max)

0.22 0.25 0.35 1.60 0.95 7.16 2.67 1.78 g.cm-³ Poorly graded mm mm mm sand

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Chapter 6

General conclusions and recommendations

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6.1. Introduction

Biocementation through ureolysis-drive MICP pathway has undoubtedly become a new soil stabilization technique which has the potential to compete with conventional methods. Biocementation involves biochemical processes that lead to the production of cement or binding material (Duraisamy, 2016). The study described in this thesis focused on investigating the efficacy to scale-up the production of S. pasteurii cells using cost-effective technical-grade reagents for soil biocementation. An important factor that determines the use of microbial enzymes in a technological process is their expense (Chaplin & Bucke, 1990). Urease often used for MICP applications are produced extracellularly from microbial cells and do not need to undergo extraction or purification.

Since ureolysis-driven MICP process requires bacterial cells for soil biocalcification soil, it is therefore imperative to ensure that the high bacterial cultivation cost is addressed. This will help resolve the economic drawback affecting biocementation and extensively improve the industrial application. While many studies have focused on other aspects affecting biocementation (i.e. homogeneity of CaCO3 and strength) (Cheng & Cord-Ruwisch, 2012; Harkes, van Paassen, Booster, Whiffin, & van Loosdrecht, 2010; Sharma & Ramkrishnan, 2016; Soon, Lee, Khun, & Ling, 2014), only a few studies investigated ways to reduce the operating costs for MICP which focuses on bacterial cells cultivation and soil treatment. However, some researchers worked on replacing the standard cultivation media with inexpensive protein-based substrates (i.e. chicken manure, corn steep liquor, lactose mother liquor and Vegemite) (Achal, Mukherjee, Basu, & Reddy, 2009; Cheng, 2012; Whiffin, 2004; Yoosathaporn et al., 2016), while others explored the use of seawater, eggshells, limestone from aggregate quarries, calcareous sand, feed and calk powders as alternative reagents to the costly analytical-grade reagents for soil biocementation treatment (Cheng, Shahin, & Cord- Ruwisch, 2014; Choi, Wu, & Chu, 2016; Ivanov, Stabnikov, & Kawasaki, 2019; Liu et al., 2017). Furthermore, only limited studies in the literature have explored the use of custom-built reactors and under non-sterile conditions to potentially lower scale-up cultivation cost of ureolytic bacteria (Aoki et al., 2018; Cheng & Cord-Ruwisch, 2013; Whiffin, 2004). Hence, it was imperative to explore possible alternative reagents that would significantly lower the cultivation cost that allows large-scale production and in-situ biocementation experiments.

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The research reported in this thesis was performed in three phases as shown in Chapter 3, Chapter 4 and Chapter 5 which were in line with the main aim of this study. S. pasteurii NB28(SUTS) strain was used throughout this study and was previously isolated by Omoregie et al., (2017) from Sarawak limestone cave sample. Contributions arising from this thesis which aid in enhancing knowledge in the area of ureolysis-drive MICP technology comprises of (i) using food-grade yeast extract as an alternative to laboratory-grade medium for low-cost cultivation of S. pasteurii; (ii) replacing analytical-grade cementation reagents with inexpensive technical-grade reagents for biocement treatment; and (iii) using custom-built stirred tank reactor to scale-up the production of S. pasteurii cells for in-situ soil biocementation.

6.2. General Conclusions 6.2.1. Bacterial cultivation using food-grade yeast extract medium

The first objective of this thesis was to determine the feasibility to cultivate the cells of S. pasteurii strain in an economical food-grade yeast extract medium and investigate its effect on bacterial production, urease activity and biocalcification. Generally, a laboratory-grade medium such as nutrient broth and tryptic soy broth is often used for the cultivation of bacterial species. These media are rich in protein and carbon concentrations (Vollum, Jamison, & Cummins, 1970). Besides, the ingredients in these costly media are usually supplemented with a trace amount of vitamins, nitrogen, inorganic salts and minerals sources for growth or maintenance of vast bacterial species. For scale-up production of ureolytic bacterial cells, it is vital to use inexpensive media that can support the growth of ureolytic bacterial cells and high urease activity (Cuzman, Richter, Wittig, & Tiano, 2015). Utilizing pure-grade cultivation media such as yeast extract did not seem feasible since it defeats the purpose to produce inexpensive large-scale of bacterial cells. Thus, Angel Yeast Extract was selected for this study because it was affordable and could also be procured in bulk quantities. In this study (Chapter 3), the low-grade cultivation medium used to cultivate S. pasteurii was inexpensive (18 USD per 1 kg). Most bacterial cells are capable of multiplying under a wide range of conditions and the bacteriological media often used for cultivation provides the substances essential for bacterial growth, however, the media concentration and hydrogen ions concentration of the medium is vital for optimum biomass performance (Vollum et al., 1970).

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In this thesis chapter, the effect of media concentration (5 to 25 g.L-1 (w/v) and initial pH medium (5 to 11) were determined before comparing the performance of the low-cost cultivation medium with eight (nutrient broth, luria broth, tryptic soy broth, fluid thioglycollate medium, lactose broth, marine broth and yeast extract) selected laboratory-grade media. Additionally, the chemicals used in this chapter were of analytical-grade and all mediums were prepared in deionized water. This was done to determine the effect of low-cost media substrate when used to replace the conventional costly media substrate. The result showed that despite high maltodextrin content (40%) present in the yeast extract medium which came with the product from the manufacturer and not deliberately added to the low-cost cultivation medium, production of S. pasteurii cells was not hindered. The growth of the bacterial cells in FG-YE medium was lucidly similar to that of LG-YE in terms of growth pattern. Further analyses showed that 15 g.L−1 media concentration and initial pH medium of 8.5 favoured optimum biomass and urease activity for S. pasteurii after being incubated at 30°C for 24 h with shaking (130 rpm).

High level of urease activity is an essential key factor for successful biocementation process (Al-Thawadi, 2008). The fact that this inexpensive yeast extract which is typically sold for food and bakery purpose was able to produce high rate urease activity and biomass concentration comparable with those of the costly microbiological media indicated that ureolytic bacterial cells could be scale-up inexpensively for CaCO3 precipitation and soil

biocementation. Since CaCO3 formation and polymorphs induced by ureolysis can be influenced by growth media, XRD analysis after biomineralization test was performed. The XRD results for the precipitate samples obtained from cementation solutions which contained laboratory-grade media (nutrient broth, tryptic soy broth and yeast extract) were identified as calcite. On the other hand, the biomineralization sample from cementation solution containing food-grade yeast extract showed the presence of calcite (94.9%) and vaterite (5.1%) polymorphs. However, there was no significant difference in the concentrations of calcite among all tested samples. Ultimately, the work in this study (Chapter 3) demonstrated the utilization of food-grade yeast extract was able to reduce the bacterial cultivation cost up to 99.80% and successfully induce calcite polymorph which is often preferred for soil stabilization. Therefore, the food-grade yeast extract media is recommended as a suitable alternative cost-effective cultivation medium for cultivation of ureolytic bacteria.

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6.2.2. Soil biocementation using technical-grade cementation reagents

The second objective in this thesis was to evaluate the feasibility of using commercially available and inexpensive technical-grade reagents to replace the costly analytical-grade reagents for bacterial cultivation and soil biocalcification. Synthetic cementation reagents (i.e.

urea and calcium chloride) are commonly used to induce CaCO3 precipitates during the MICP process. Correspondingly to the laboratory-grade substrate, most researchers also use analytical-grade cementation reagents for MICP experiments because they are of high-purity. Unfortunately, these reagents are too costly for industrial MICP applications (Zhu & Dittrich, 2016). To reduce this cost issue affecting MICP technology, this study (Chapter 4) was carried to determine the likelihood of using low-grade reagents for bacterial cultivation and soil biocalcification.

S. pasteurii cells were grown in two different types of cultivation medium (analytical- grade and technical-grade) prepared in both tap water and deionized water. After studying their biomass and urease activity performances which showed the comparable outcome, MICP treatment on sandy soil was performed using the bacterial cells and cementation solutions. To ensure the low-grade reagents could produce comparable results, 0.25-1.0 M concentrations of the cementation reagents prepared in tap water and deionized water were used. Furthermore, the bacterial cells cultivated were also used applied to the sand for ureolysis and formation of nucleation site with respective ingredient purity (high-grade or low-grade). The result showed for bacterial growth and pH profiles were comparable irrespective of which medium they were cultivated. The biomineralization test of cementation solutions prepared using technical-grade and analytical-grade reagents in tap water showed visual differences in the rate of precipitation. It took 1 h for the precipitates to settle at the bottom of the flasks for sample in technical-grade cementation solution and 4 h in the analytical-grade cementation solution. The XRD analysis of the precipitates showed no significant difference and had undistinguishable polymorphs

peaks and calcite was identified as the dominant CaCO3 polymorph. Among all CaCO3 polymorph (calcite, vaterite and aragonite), calcite is often regarded as the preferred choice for biocementation because of its stability and ability to improve soil stabilization better than other

CaCO3 polymorphs (Dhami, Mukherjee, & Reddy, 2016). Although the formation of vaterite and calcite has shown not to impede soil solidification after biocement treatment (Dhami et al., 2017).

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Furthermore, surface percolation was selected as soil MICP treatment method because of it is easy to use, does not require heavy machinery, utilizes normal gravitational force for a cascade of the solutions into the sand column and have been shown to suitable for soil at the depth of 2 m (Cheng & Cord-Ruwisch, 2012; Cheng, Shahin, & Cord-Ruwisch, 2017; Mujah, Shahin, & Cheng, 2017). The PVC column (height of 11.8 cm and diameter of 5.8 cm) was used to house untreated sand in this study. pH measurements of the effluent samples obtained after biocement treatments indicated comparable ureolysis rate for both the samples treated with analytical-grade solutions (7.39 ±0.01 to 9.31 ±0.02) and technical-grade solutions (7.44 ±0.05 to 9.26 ±0.02). This meant the exogenous bacterial cells could enable the similar rate of urease activity irrespective of purity level for the reagents used for biocementation. It is an accepted phenomenon that increases in CaCO3 contents of the biocemented samples correlate with the increase in strength for improved sand solidification. This was the same outcome in this study for samples with high-grade and low-grade cementation solutions. As expected, samples treated with 0.25 M of the cementation solution had the lowest CaCO3 contents and

surface strength, while those treated with 1.0 M of cementation solution had the highest CaCO3 contents and surface strength. During biocementation, clog formation (white precipitation on sand columns) occurred to samples treated with higher concentration. Such occurrence is often due to rapid ureolysis and precipitation reactions during solution transportation (Gomez, Graddy, DeJong, & Nelson, 2019). It is recommended to use a high concentration of cementation reagents on soil which targets improved strength or stiffness in the surface layers.

However, if more CaCO3 are needed at lower soil layer, then the lower high concentration of

cementation reagents should be applied to prevent excess CaCO3 formation or wastage at the top layer of the soil specimens. The SEM observations were only performed on samples treated with 1 M cementation solution. Ureolytic bacterial cells in the presence of a high concentration of cementation solution can result in a spherical or rhombohedral crystal which could generally be calcite and vaterite (Al-Thawadi, 2008). The SEM micrographs suggested there were no

significant differences among the morphological structures of the CaCO3 crystals formed on the surface of the treated sand particles. However, there were noticeable differences shown in the topographical features of CaCO3 crystal precipitates. This study was able to show the efficacy of using technical-grade reagents for bacterial cultivation and biocement treatment prepared in both tap water and deionized water. Cost analysis indicated that for soil treatment with analytical-grade reagents (3.33-13.29 US$/L) and technical-grade cementation reagents (0.07-0.26 US$/L), there was a cost difference of 47 to 51-fold.

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6.2.3. Large-scale bacterial cultivation using custom-built reactor

The third objective in this thesis was to investigate the feasibility to scale-up the production of S. pasteurii cells from small-scale to large-scale using a custom-built stainless steel stirred tank reactor (3 m3) under non-sterile conditions and determine its suitability for soil biocementation. Scale-up production of the ureolytic bacterial cells for in-situ biocementation are costly. In most cases, chemostats (1-5 L) and shake flasks are often used to cultivate various ureolytic bacterial species (Cheng & Cord-Ruwisch, 2013; Işik et al., 2010; Kalantary & Kahani, 2019; Omoregie et al., 2017). However, scalability of bacterial cells for field-scale experimentations will require reactors which are in several orders of magnitude greater than laboratory-scale (Zhu & Dittrich, 2016). Large-scale production of the microorganism is susceptible to an increase in the cultivation cost and drive up the total cost of biocementation. Few researchers have shown that cultivation of ureolytic bacterial cells in the custom-built reactor in small-scale and pilot-scale under non-sterile condition can help minimize the cultivation.

Custom-built stainless steel stirred tank reactor was used to investigate the feasibility to scale-up the cells of ureolytic bacteria to a final volume of 2400 L and determine its biocalcifying ability for in-situ biocalcification. S. pasteurii NB28(SUTS) used in preceding chapters (Chapter 3 and 4) was also used in this current study (Chapter 5) to serve as inoculum for the outdoor experiments. These inoculum cells (65 L) were prepared under the sterile laboratory-scale condition to avoid any intricacy or complication that might affect determining the origin of any bacterial contamination that might occur during bacterial scalability on site. Scalability of the bacterial cells was carried out from 216 L (small-scale cultivation) to 720 L (pilot-scale cultivation and 2400 L (large-scale cultivation) using the custom-built reactor. The desired working volume (2400 L) was selected as a final scale-up phase because it was required to perform an in-situ biocement treatment for a 10 m3 sandbox which was to performed by a member of our research team (Wani Senian, Civil Engineering PhD Candidate). During the fermentation periods, the performance (OD, pH, urease activity, DO, TDS and salinity) of the bacterial cultures were monitored and the presence of any contaminating species were identified. Pivotal to this study was the efficacy of the bacterial cells cultured to large-scale under non-sterile condition be capable of enhancing the stability and durability of granular soil.

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Upon completion of in-situ soil biocement experiment using the cultured bacterial cells

and technical-grade cementation solution, the pH effluent, CaCO3 contents and microstructural analyses were performed. Growth kinetics (1st rate of reaction) is pivotal for the implementation of MICP (Jain & Arnepalli, 2019). Hence, the biomass, pH and urease activity were carefully monitored throughout the scale-up process. At the end of the 90 h cultivation period, the maximum OD for 216 L, 720 and 2400 L seed cultures were 0.5 ±0.00, 1.6 ±0.02 and 1.8 ±0.09, respectively. The measured urease activities were 9.3 ±0.14 mM urea hydrolysed.min−1 for 216 L seed culture, 11.1 ±0.13 mM urea hydrolysed.min−1 for 720 L seed culture and 11.1 ±0.48 mM urea hydrolysed.min−1 for 2400 L culture seeds.

Culture samples collected from the custom-built reactor showed that there were four ureolytic isolates denoted as OA6 (SUTS), OA7 (SUTS), OA10 (SUTS) and OA18 (SUTS), respectively. The 16S rRNA gene sequencing successfully identified all the ureolytic bacterial isolates as Pseudogracilibacillus marinus. The DNA sequences of the isolates have been deposited in The National Center for Biotechnology Information GenBank database having accession numbers MK595743, MK595744 MK595745 and MK595747. It is not entirely known how and why contamination occurred during the non-sterile cultivation period. Before use, the custom-built reactor was cleaned using liquid detergent and 5% sodium hypochlorite. Monitoring observations suggested that the contamination occurred during 2400 L scale-up phase. This observation was contrary to the study carried out by Whiffin (2004) who reported the absence of no contaminating species during the pilot-scale cultivation of S. pasteurii under non-sterile condition. However, this possible due to the presence of high urea and ammonia concentrations and pH conditions ensured throughout the cultivation period. Ureolysis-based MICP is known to be a soil modification method employed for increasing the strength or reducing the permeability of soil (Gao, Tang, Chu, & He, 2019). Hence, it was essential to determine if ureolysis rate and biocalcification effect was still feasibility during MICP soil treatment or probably the presence of contaminating species might have aggravated the CaCO3 enactment.

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The in-situ soil biocementation carried out showed that the bacterial cells were able to promote biocalcification and cementation of sand columns after the treatment. Each set of columns were augmented with bacterial cells only on the first day of the treatment while the cementation solutions were percolated into the soil for different treatment cycles (3 days, 6 days and 9 days). All the soil columns showed successful soil solidification after 8 weeks

curing period. The distribution of CaCO3 at the top, middle and bottom layers of the treated samples showed the successful outcome of biocementation. As expected the top layers of all

soil columns had the highest amount of CaCO3. Also, soil columns treated for 9 days with

cementation solution had the highest amount of CaCO3 compared to samples treated with 3

days and 6 days. Microstructural and pH effluent analyses confirmed CaCO3 crystal formations and urease hydrolysis rate. The study carried out in Chapter 5 of the thesis suggested that it is possible to scale-up ureolytic bacterial cells using a custom-built stirred tank reactor and under non-sterile conditions. The cost analysis showed that the 3m3 reactor was fabricated for USD 6574.97 and the cost of ingredients used to scale-up the bacterial cells ranged from USD 94.49 to 1328.46. This work was successful in showing that it is feasible to reduce the cultivation cost of ureolytic bacteria and has the potential to be utilized for field-scale experiments. However, the limitations experienced during the scale-up process should be addressed before using the custom-built reactor for field-scale application to reduce any possible bacterial contamination which might occur during the scale-up process and increase the biomass production.

6.3. Recommendation for future research

6.3.1. Utilizing low-cost cultivation medium

The proposed food-grade yeast extract medium was found suitable for cultivation of ureolytic bacterial cells. Future studies should explore this alternative medium because it can help to lower bacterial cultivation cost. The continuous dependence on laboratory-grade media will only further limit the chances to scale-up ureolytic bacterial cells for MICP field-scale experiments. This food-grade yeast extract can be used to cultivate a wide range of bacterial species as shown in Table 6.1 which are essential for a wide range of biotechnological applications. To test the potential usage of this media selected beneficial bacterial strains were cultivated in food-grade yeast extract medium (20 g.L-1) under sterile laboratory-conditions and incubated at 32oC for 24 h with shaking (150 rpm).

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The preliminary findings suggest that this inexpensive medium can also be used for cultivation other types of bacterial species. Further studies should be carried out to determine how optimized conditions can favour higher biomass or enzyme production. More work could be carried out to determine the suitability of this medium on the growth of these beneficial microorganisms for future applications.

Table 6.1: Different bacterial species are grown in the food-grade yeast extract medium

Bacterial species Strain OD600 Sporosarcina pasteurii DSM 33 1.283 Bacillus megaterium ATCC 14581 1.229 Bacillus cereus DSM 31 0.907 Bacillus subtilis ATCC 6051 1.724 Escherichia coli ATCC 29425 1.503 Salmonella typhimurium DSM4883 1.658 Klebsiella pneumoniae ATCC 24735 1.430 Streptococcus pnuemoniae ATCC 55143 1.669 Pseudomonas aeruginosa ATCC 15442 1.673 Staphylococcus aureus ATCC 25923 1.767

6.3.2. Durability test of biocemented soil specimens

Almost all MICP treated sand specimens are subjected to several days or weeks monitoring studies. Most studies on environmental factors (i.e. ammonium concentration, chemical reactants, the concentration of bacterial cells and oxygen availability) affecting MICP process or soil biocementation are carried out under in-vitro or regulated conditions. Environmental factors play key roles in the durability of biocementation. However, studies on the durability of biocementation especially for the long-term effect are not common in the literature. It is worthwhile to investigate the long-term effect or changes that might occur to the mechanical properties of the biocemented samples. External environmental factors such as atmospheric temperature, relative humidity, moisture and climate condition are known to have the capacity of affecting building and construction materials.

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For example, exposure of concrete to high or abrupt atmospheric temperature change can increase the rate of evaporations which in turn can affect its compressive strength or lead to cracking. Excessive rainfall can affect the porosity or permeability of treated soil samples. More so, biochemical reactions from microorganisms may also cause degradation or deterioration to building materials. Hence, it seems pivotal for future MICP studies to carry out more outdoor experiments on soil biocementation to mimic real site work. Evaluation of the effects of environmental conditions, if any, on the compressive strength, stiffness, permeability, calcite content should be investigated.

Fig. 6.1: Digital image of biocemented sand specimens after treatment (6 months).

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Fig. 6.2: Digital image of biocemented sand specimens subjected to prolong outdoor environmental conditions. Samples after 12 months (A) and 24 months of biocementation treatment.

Future soil biocementation work need to move away from bench-scale to field-scale studies, even small-scale outdoor experiments which show exposure to environmental condition is a good start and will help fill the knowledge gap in the literature. It would be interesting to know how biocemented soil specimens perform after being subjugated to prolong outdoor curing duration after in-situ treatment. These specimens could be examined for 12-24 months with monthly interval analysis and compared with samples being cured indoor. The effects of environmental conditions on the samples treated outdoor would be able to show the durability of biocementation after prolong curing period. Such studies would be able to provide important insight into the suitability of this technology for commercial implementations and provide essential information which could be used to improve biocementation.

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Samples of the biocemented soil (Fig 6.1) performed in Chapter 5 of these studies were subjected to preliminary examination through visual observation. The samples were placed outdoor and exposed to natural environmental conditions as shown in Fig. 6.2. Malaysia is a tropical rainforest climate region experiences frequent downpour of rain and high humidity throughout the year. Also, the atmospheric temperature of the localized province (Kuching, Sarawak) ranges from 22oC to 36oC. These conditions were considered to be suitable for the preliminary durability test. The specimens were undisturbed throughout the observation period (24 months). Visual observation in Fig. 6.2 showed vivid physical changes which had occurred to the samples. It appears to prolong exposure to environmental conditions that led to an improvement in the top layers of the soil specimens. The samples seemed to have enhanced solidification since being kept outdoor for 24 months. However, the middle and bottom regions of the specimens did not seem to have improved solidification. Future work has to be performed to explain this outcome.

6.3.3. Ammonia production

Biocementation is seen as a new technology which has helped changed modern construction and building industry. The use of beneficial microbes to help improve the mechanical properties of soil has drawn enormous attention from research and industrial communities. Despite numerous potential and on-going applications, the production of ammonia gas which occurs during ureolysis process limits the implementation of this technology. Ammonia production during soil biocementation occurs as a result of ureolysis- driven MICP process. The hydrolysis of urea by ureolytic bacteria allows the generation of 2 moles of ammonium for each urea hydrolysed. This later leads to the production of ammonia gas. Ammonia vitalization which is the generation of ammonia gas at alkaline pH as a result of nitrogen loss from soil surface can also occur during soil biocementation. The pungent smell during soil biocementation is a good indicator for active ureolysis process by the beneficial microbes, however, the release to the atmosphere is of great concern. Researchers need to take ensure ammonia gas production during in-situ biocementation of soil are not in high concentration because it can be detrimental to human health and the environment. More future researches should be performed to increase the field-scale implementation of MICP technology and make it ecologically-friendly.

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There are certain compounds which can be used to temporarily hinder urea hydrolysis and thus slowing down the rate or ammonia gas production. Since ureolysis process is pivotal for

CaCO3 precipitation, as a suggestion, to avoid any alteration or intricacy that might ensue during biocementation process, urease inhibitors should be applied on surfaces of treated samples upon completion of MICP treatment. For ureolysis, high pH is favourable for biocementation, hence application of urease inhibitors during bacterial cultivation or soil biocementation may complicate or affect the stabilization or durability of the biocemented soil product. Future research can explore the use of ammonium thiosulfate, an inorganic compound to possibly reduce ammonia gas production. Ammonium thiosulfate can be procured commercially at the bulk quantity and low-cost. There are other compounds which can be used as urease inhibitors but researchers have to ensure they are not toxic, have chemical stability and can be easily procured at low cost. Inorganic adsorbents such as activated carbon, alumina, silica gel and gypsum can be investigated for their suitability in trapping ammonia gas during biocementation process. Future studies should aim to understand the biochemical process that occurs when materials such as urease inhibitors and inorganic adsorbents are used as reagents for soil biocementation. It will be helpful for the researcher to find out how these materials may help trap ammonia gas or interfere in the biocementation process. Further research can also look into the ammonia gas adsorption capacities of the materials being explored and ensure the materials used have high adsorption capacities.

It is imperative to note that biocementation is not a replacement for conventional cement and not yet a comprehensive environmentally-friendly technology. However, some investigations have been demonstrated in the literature to show that MICP has the potential to become a complete environmentally-friendly. For this to occur, certain isues need to be addressed. Urea is often used in MICP experiments to allow S. pasteurii produce urease and

induce CaCO3 precipitation. This ensures that the engineering properties of soil results in enhanced strengths and stiffness. However, for MICP to be regarded as a complete environmentally- friendly technology, the dependence on urea for urease production need to be minimized. Urea which is commonly used as a constituent of cementation solution and growth media for biocementation process and bacterial cell propagation, unfortunately, has a

large greenhouse footprint. Most of the CO2 used to manufacture urea comes from CO2

generated during the production of ammonia, thus responsible for the high CO2 content of GHG emissions (Shi et al., 2020).

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The global carbon footprint of technical-grade urea fertiliser ranges between 1.484 to -1 3.002 CO2eq.kg product (including CO2 captured in the product) (Antione & Bjarne, 2019).

It was reported in 2015 that 0.7 tonnes of CO2 are required to produce approximately 1 tonne of urea (Dobrée, 2016). However, the total carbon footprint derived through a full life-cycle analysis is 5.15 tonnes CO2-equivalent (Brown, 2016). Since urea is vital global food production, the fertilizer industries and researchers have focused on how to minimize the environmental impact associated with urea production. This has resulted in implementing new approaches that can help mitigate energy saving and GHG emissions which includes using renewable feedstock and methane for production of green urea, replacement of old melting/prilling process with the newest technology for urea granulation, and integration of

surplus renewable energy to power the electrolytic generation of H2 (Alfian & Purwanto, 2019; Driver et al., 2019; Khan, Al-Awadi, & Al-Rashidi, 2016).

Consequently, urea production which uses conventional process may be halted as fertilizer industries adopt new environmentally-friendly methods. Nonetheless, several researchers have reported alternatives sources to synthetic urea for MICP process Cuzman et al., (2015) suggested that since urea can be found abundantly in the urine and faeces, they should be considered for future biocementation studies. Subsequently, recent investigations on human and pig urine samples serving as an alternative source of urea for MICP applications were reported by Chen et al., (2018) and Lambert & Randall, (2019). These scholars were able to successfully substitute analytical-grade and industrial-grade urea with urine and produce biocement bricks/columns. Their findings also show that MICP cannot only become a green sustainable technology, it can also be used for recycling of waste materials and mitigate environmental pollution. Future researchers can also consider other alternatives as sources of urea for MICP experiments. Nitrogen sources which may be investigated as a replacement to urea for MICP process are inorganic fertilizers (i.e. ammonium sulphate, calcium ammonium nitrate, urea-ammonium sulfate, liquid urea-ammonium nitrate, environmentally smart nitrogen) and organic fertilizers (i.e. animal manure and slurry, industrial wastewater, composts and sewages).

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6.3.4. Improving the custom-built reactor

Future studies can adopt the use of a single custom-built reactor for economic scale-up of bacterial production. However, the modification should be carried out to improve the production process and biomass yield. The work performed in Chapter 5 of this thesis was completely done under non-sterile environment except for the S. pasteurii inoculum which was cultivated in the laboratory (under sterile-condition). Surely, the presence of bacterial contaminant during the scale-up process in the custom-built reactor could compromise the growth of ureolytic bacteria due to competition for the consumption of nutrient and dominance. Hence, it would be essential for a future custom-built reactor to have systems that minimize contamination or support sterilization. Also, in a situation where a high concentration of urea is needed during bacterial cultivation which also helps to reduce non-beneficial microbes, outlet pipes can be installed in the reactor that allows collection of ammonia gas produced during ureolysis process. This pipes can be connected to a simple custom-built device that acts as a wet scrubber for removal of pollutant gas. The accumulation of the ammonia gas during cultivation, if no air outlet is present can be toxic for the bacterial cells during cultivation and result in lower biomass production. 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. Hence, there the custom-built reactor had no sterilization feature. The intention for this was to ensure that during in-situ implementation, bacterial cell propagation done under non-sterile conditions, would not hinder its performance. Most conventional MICP applications are done using sterile cultivation medium and process to grow the bacterial cells, but in the real world situation, it is not necessary to use sterile conditions because it will only add up to the bacterial production cost. Therefore, future studies should investigate how MICP performance could be optimized under non-sterile and in-situ conditions.

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6.3.5. Economic feasibility of biocementation Construction biotechnology has increasing become a promising area of discipline which aims to produce building materials that result in low or no carbon footprint and environmental pollution. Biocementation has the potential to penetrate the commercial market because it can be used to produce construction materials which require low temperature and renewable energy sources (Myhr et al., 2019). However, this technology is expensive for scale-up and field-scale implementation. Nevertheless, there are several companies like BioCement Technologies, Biomason and Bachy Solentanche that are utilizing biocementation technology for commercial engineering applications such as bacteria-based additive to topsoil for prevention of erosion, bioconcrete production soil stabilization. Presently there is no lucid overall economic feasibility of biocementation in real-world practice which also affects the potential niche application where this technology can outperform other existing alternative technologies. Two niche areas where MICP technology gains fervent competitiveness with other technologies are in bioconcrete repair or production and biocement treatment of fine-grain soils. For biocementation to be accepted into the commercial market, it needs to show it is relatively cheaper or similar to the existing alternatives. Although, for the economic feasibility of biocementation to succeed, two important criteria needs to be addressed, namely, (i) minimizing the bacterial cells production cost and (ii) minimizing the cementation treatment cost (Kakelar, Ebrahimi, & Hosseini, 2016). It has also been suggested that by augmenting and stimulating indigenous ureolytic microorganisms through injection method, it may eliminate the need for bacterial cultivation and reduce the treatment cost (Gomez et al., 2019).

Interestingly, a recent review paper by Rahman et al., (2020) presented a detailed cost assessment and environmental benefit of MICP technology using three scenarios (only paver blocks treated with MICP, pavers and sub-base layer treated with MICP, and pavers and subgrade treated with MICP). The total cost of chemicals used to produce treatment solutions per the mix designs was presented in Australian $. The estimated cost of the treatment reagents based on current market price (industrial-grade) used in this review paper was based on a report by Omoregie, Ngu, Ong, & Nissom, (2019). The first study scenario (only paver blocks treated with MICP) which was reported by Achal, Mukherjee, & Reddy, (2011) showed that to treat 1 m3 of concrete requires approximately 600 L of treatment solution. This resulted in a treatment cost of approximately $4.5 per m3 for the MICP process.

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The calculated assessment (total economic and environmental costs) by Rahman et al., (2020) showed that for MICP treatment of pavers on a road (1 km), it will be 25% less expensive than conventional concrete pavers and contributes to a 19% environmental benefit

(about 47,000 kg of CO2). While, the second study scenario (pavers and sub-base layer treated with MICP) reported by Porter, Dhami, & Mukherjee, (2018) used cementation solution of 250 L with 20 treatment cycles and bacterial solution (11% by cell mass). This resulted in a treatment cost of approximately $215 per m3 for the MICP process. The assessment by Rahman et al., (2020) indicated that for a 1 km road treatment using MICP technology, the total cost will be 1.6 times more expensive than conventional cement treatment and result to a 3.4 times environmental impact. The third study scenario (pavers and subgrade treated with MICP) reported by van Paassen et al., (2010) demonstrated a large-scale application of MICP on a 3 3 3 sandbox (100 m ). They injected 5 m of nutrient broth with bacterial cells and 5 m of CaCl2 solution (0.05 M) and followed by 96 m3 injection of the treatment solution. This resulted in a treatment cost of approximately $60 per m3 for the MICP process which is comparable with scenario 1. The calculated assessment by Rahman et al., (2020) indicated that for a 1 km road treatment using MICP technology, the total cost results in 9% economic loss and about a 19%

environmental loss (in the form of CO2) when compared with conventional cement treatment.

Hence, future scholars should consider include the total cost materials or reagents used during the MICP treatment. Also, they should consider including information on the overall economic benefits and environmental impact of their respective studies. This will help improve the knowledge required to assess the economic feasibility and environmental sustainability of MICP technology. Also, future research should provide or determine quality control and quality assurance of ureolysis-driven MICP as a standard process for industrial application. The implementation of an accepted standard will provide consistency and required outcome or expectation of MICP process. Also, the published standard process of the MICP for industrial application will help provide indispensable information and benchmark data which others can use to improve the quality of their MICP products. Things which should be included in quality control and quality assurance of ureolysis-driven MICP process should include the concentration of cementation reagents, compressive strength, curing duration, the specific activity of urease enzyme, the concentration of bacterial cells and calcite contents. The standard values from these factors can therefore be used as baseline cases.

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6.4. Summary The MICP through the facilitation of microbial catalyst is a technology that can be used for numerous promising biotechnological and engineering applications. Biocementation technology for the past 25 years has shown it can serve as an alternative candidate to improve the engineering properties of soil. However, conventional MICP technology has been hindered from becoming commercially attainable and competitive with existing technologies due to its uneconomic feasibility. The findings from this thesis have shown that inexpensive technical- grade reagents can comparatively perform soil biocement treatment with costly analytical- grade reagents and be used for bacterial cell propagation. More so, the scale-up process of ureolytic bacterial cells could be performed using a custom-built reactor under non-sterile condition. As a final point, this thesis further stresses the need for future research to focus more on experimenting with low-cost reagents so MICP implementation reduced.

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Sharma, A., & Ramkrishnan, R. (2016). Study on effect of microbial induced calcite precipitates on strength of fine grained soils. Perspectives in Science, 8, 198–202. doi:10.1016/j.pisc.2016.03.017

Shi, L., Liu, L., Yang, B., Sheng, G., & Xu, T. (2020). Evaluation of industrial urea energy consumption (ec) based on life cycle assessment (lca). Sustainability, 12(9), 3793.

229

van Paassen, L. A., Ghose, R., van der Linden, T. J. M., van der Star, W. R. L., & van Loosdrecht, M. C. M. (2010). Quantifying biomediated ground improvement by ureolysis: large-scale biogrout experiment. Journal of Geotechnical and Geoenvironmental Engineering, 136(12), 1721–1728. doi:10.1061/(ASCE)GT.1943-5606.0000382

Vollum, R. L., Jamison, D. G., & Cummins, C. S. (1970). The cultivation of bacteria.In fairbrother’s textbook of bacteriology (10th ed., pp. 32–41). Oxford: Butterworth- Heinemann. doi:https://doi.org/10.1016/B978-0-433-10100-0.50008-X

Whiffin, V. S. (2004). Microbial CaCO3 precipitation for the production of biocement. Doctoral Thesis, Murdoch University. Perth, Australia.

Zhu, T., & Dittrich, M. (2016). Carbonate Precipitation through Microbial Activities in Natural Environment, and Their Potential in Biotechnology: A Review. Frontiers in Bioengineering and Biotechnology, 4, 1–21. doi:10.3389/fbioe.2016.00004

230

Appendix A

Permission from the copyright owner

231

From: Researcher Support To: Armstrong Ighodalo Omoregie Subject: Re: Permission to use article materials in my PhD thesis [191107-006598] [191107-006598] Date: Friday, 8 November, 2019 4:58:30 PM

Dear Dr Omoregie,

DOI: 10.1016/j.conbuildmat.2019.116828. Title: Biocementation of sand by Sporosarcina pasteurii strain and technical-grade cementation reagents through surface percolation treatment method”, Construction and Building Materials

DOI: 10.1016/j.bcab.2018.11.030 Title: Low-cost cultivation of Sporosarcina pasteurii strain in food-grade yeast extract medium for microbially induced carbonate precipitation (MICP) application

Thank you for your e-mail.

I can confirm that authors can use their articles, in full or in part, for a wide range of scholarly, non-commercial purposes one of which is inclusion in a thesis or dissertation.

See the following link for further information on this https://www.elsevier.com/about/our-business/policies/copyright/personal-use

As you will be able to see from the above link, our policy is to allow authors to use their work in their thesis or dissertation (provided that this is not to be published commercially).

Therefore, I can confirm that you can make it publicly available on your university web site/repository for non- commercial use.

If you require further clarification please contact [email protected] who will be able to help

I hope this information is of assistance, please do not hesitate to contact me if you have any questions.

Kind regards,

Cristabel Enaron Researcher Support ELSEVIER

Find out some simple ways to share your research data, including features that are directly available when you submit your research article to an Elsevier journal.

For assistance, please visit our Customer Support site where you can search for solutions on a range of topics and find answers to frequently asked questions.

From: Administrator Date: 07/11/2019 05.06 AM Dear Customer,

Thank you for your enquiry.

Ticket number 191107-006598 has been opened on your behalf and we aim to respond within 24 hours (excluding weekends).

Please use this ticket number in any future correspondence and please do not change the subject line of this email when you reply.

For further support information please visit our ScienceDirect Support Center.

Regards,

Elsevier Online Solutions

From: Armstrong Omoregie Date: 07/11/2019 05.06 AM

Hello,

I am a final year PhD student at Swinburne University of Technology Sarawak Campus and I will be submitting my thesis for examination very soon.

The following research articles are part of my doctoral study and they have been published in Elsevier journals:

1. Omoregie, A. I., Palombo, E., Ong, D. E. L., & Nissom, P. M. (2019), “Biocementation of sand by Sporosarcina pasteurii strain and technical-grade cementation reagents through surface percolation treatment method”, Construction and Building Materials. 228C, 116828. 10.1016/j.conbuildmat.2019.116828.

2. Omoregie, A. I., Hei, N. L., Ong, D. E. L. & Nissom, P. M. (2019), “Low-cost cultivation of Sporosarcina pasteurii strain in food-grade yeast extract medium for microbially induced carbonate precipitation (MICP) application”, Biocatalysis and Agricultural Biotechnology, 17, 247-255. https://doi.org/10.1016/j.bcab.2018.11.030.

Hence, I am seeking permission to include these materials as Chapter 3 & Chapter 4 in my thesis for examination.

Am I allowed to do so? If more information is needed, please let me know.

Kind Regards

This communication is confidential and may be privileged. Any unauthorized use or dissemination of this message in whole or in part is strictly prohibited and may be unlawful. If you receive this message by mistake, please notify the sender by return email and delete this message from your system. Elsevier B.V. (including its group companies) shall not be liable for any improper or incomplete transmission of the information contained in this communication or delay in its receipt. Any price quotes contained in this communication are merely indicative and may not be relied upon by the individual or entity receiving it. Any proposed transactions or quotes contained in this communication will not result in any legally binding or enforceable obligation or give rise to any obligation for reimbursement of any fees, expenses, costs or damages, unless an express agreement to that effect has been agreed upon, delivered and executed by the parties.

[---001:003663:41074---] From: [email protected] on behalf of [email protected] To: Armstrong Ighodalo Omoregie Subject: Case #00927086 - Permission to use article materials in my PhD thesis [ ref:_00D30oeGz._5000c1vFYJz:ref ] Date: Wednesday, 13 November, 2019 12:31:22 AM

Dear Armstrong Omoregie,

I hope you are having a wonderful day! Thank you for contacting Copyright Clearance Center's RightsLink Service, my name is Milan and I'd be happy to assist you today.

I've looked into these two articles for you and it appears that permissions can be obtained through our services. One would need to go through RightsLink, while the other one can go through our Marketplace.

For the one titled "Biocementation of sand by Sporosarcina pasteurii strain and technical- grade cementation reagents through surface percolation treatment method", please start HERE. This will take you to the RightsLink page where you will be able to make your request. Simply follow the process, and let me know if there's anything confusing that I could help you with.

For the second one titled "Low-cost cultivation of Sporosarcina pasteurii strain in food- grade yeast extract medium for microbially induced carbonate precipitation (MICP) application", you can start HERE. The process is a bit different, so if you run into any issues, please let me know.

I hope I was able to answer your question! Should you have any further concerns or issues, do not hesitate to reach out to me anytime. I wish you the best of luck with your thesis!

Kind regards,

Milan

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------Original Message ------From: Armstrong Ighodalo Omoregie [[email protected]] Sent: 11/7/2019 6:03 PM To: [email protected] Subject: Permission to use article materials in my PhD thesis

Hello,

I am a final year PhD student at Swinburne University of Technology Sarawak Campus and I will be submitting my thesis for examination very soon.

The following research articles are part of my doctoral study and they have been published in Elsevier journals:

Hence, I am seeking permission to include these materials as Chapter 3 & Chapter 4 in my thesis for examination.

Am I allowed to do so? If more information is needed, please let me know.

Kind Regards,

ARMSTRONG I. OMOREGIE PhD Candidate (Applied Biotechnology)

Centre for Sustainable Technologies

11/14/2019 https://marketplace.copyright.com/rs-ui-web/mp/license/1215cbf5-cd0f-4d1d-9301-75fd1ebd7e1a/20d25de5-d61f-4cfd-8303-129a4…

Elsevier Science & Technology Journals - License Terms and Conditions

Order Date 13-Nov-2019 Order license ID 1003506-1 ISSN 1878-8181 Type of Use Republish in a thesis/dissertation Publisher Elsevier Portion Chapter/article

LICENSED CONTENT

Publication Title Biocatalysis and Rightsholder Elsevier Science & Agricultural Biotechnology Technology Journals Article Title Low-cost cultivation of Publication Type Journal Sporosarcina pasteurii Start Page 247 strain in food-grade yeast extract medium for End Page 255 microbially induced Volume 17 carbonate precipitation (MICP) application Author/Editor International Society of Biocatalysis and Agricultural Biotechnology. Date 01/01/2010 Language English Country United Kingdom of Great Britain and Northern Ireland

REQUEST DETAILS

Portion Type Chapter/article Rights Requested Main product Page range(s) 247-255 Distribution Worldwide Total number of pages 8 Translation Original language of publication Format (select all that Electronic apply) Copies for the disabled? No Who will republish the Academic institution Minor editing privileges? No content? Incidental promotional No Duration of Use Life of current edition use? Lifetime Unit Quantity Up to 499 Currency USD

NEW WORK DETAILS

Title A feasibility study to scale- Institution name Swinburne University of up the production of Technology Sarawak Sporosarcina pasteurii, Campus using industrial-grade Expected presentation 2020-06-01 reagents, for cost-eective date in-situ biocementation Instructor name Assoc. Prof. Peter Morin Nissom

ADDITIONAL DETAILS https://marketplace.copyright.com/rs-ui-web/mp/license/1215cbf5-cd0f-4d1d-9301-75fd1ebd7e1a/20d25de5-d61f-4cfd-8303-129a434c843e 1/4 11/14/2019 https://marketplace.copyright.com/rs-ui-web/mp/license/1215cbf5-cd0f-4d1d-9301-75fd1ebd7e1a/20d25de5-d61f-4cfd-8303-129a4… Order reference number N/A The requesting person / Armstrong Ighodalo organization to appear Omoregie on the license

REUSE CONTENT DETAILS

Title, description or PhD thesis Title of the Low-cost cultivation of numeric reference of the article/chapter the Sporosarcina pasteurii portion(s) portion is from strain in food-grade yeast extract medium for Editor of portion(s) Ong, Dominic Ek Leong; microbially induced Omoregie, Armstrong carbonate precipitation Ighodalo; Nissom, Peter (MICP) application Morin; Ngu, Lock Hei Author of portion(s) Ong, Dominic Ek Leong; Volume of serial or 17 Omoregie, Armstrong monograph Ighodalo; Nissom, Peter Page or page range of 247-255 Morin; Ngu, Lock Hei portion Issue, if republishing an N/A article from a serial Publication date of 2019-01-01 portion

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Biocementation of sand by Sporosarcina pasteurii strain and technical- grade cementation reagents through surface percolation treatment method Author: Armstrong I. Omoregie,Enzo A. Palombo,Dominic E.L. Ong,Peter M. Nissom Publication: Construction and Building Materials Publisher: Elsevier Date: 20 December 2019

Please note that, as the author of this Elsevier article, you retain the right to include it in a thesis or dissertation, provided it is not published commercially. Permission is not required, but please ensure that you reference the journal as the original source. For more information on this and on your other retained rights, please visit: https://www.elsevier.com/about/our-business/policies/copyright#Author-rights

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https://s100.copyright.com/AppDispatchServlet 1/1 From: [email protected] on behalf of [email protected] To: Armstrong Ighodalo Omoregie Subject: Case #01012438 - Permission to use article in my PhD thesis [ ref:_00D30oeGz._5004Q1yoYmP:ref ] Date: Wednesday, 4 March, 2020 8:36:47 PM

Dear Armstrong Omoregie,

Thank you for contacting Copyright Clearance Center's RightsLink Service. My name is D.J., and it would be my pleasure to assist you.

Based on the information you provided I am happy to inform you that it is possible to obtain the permission to reuse your own article "A feasible scale-up production of Sporosarcina pasteurii using custom-built stirred tank reactor for in-situ soil biocementation" in your upcoming thesis and would gladly guide you through the steps that you need to take.

Please access this - LINK and you will be forwarded to our Marketplace for permissions website If the article found is the appropriate one please click on the Request Permissions link in the bottom right corner Please note you are to be prompted to a new page where you need to choose the type of use of the content from the drop down menu titled "Type of Use (TOU)" appearing underneath the journal's bibliographic details. To further help you with your order and for you to select the best option suited for the permission you require, we strongly suggest that you take a moment to read the publisher’s definition for each option in RightsLink by clicking the orange circles with the “?” and carefully review the definitions that best fits the use of the content. Please note that the selection in RightsLink vary by publisher. Upon filling out all the necessary information in all drop down menus, you will be provided with the price of the permission. However, depending on the selections you have chosen during the order process, you may be given a "Not Available" quote. This means that your order is considered as a Special Order and once completed and submitted will be reviewed by the rightsholder before you are provided with an offer that you can later accept or decline If the price is suitable please continue on entering the information in the section NEW WORK DETAILS and to proceed click on the NEXT button. Please be advised that your order will not be completed until you select CHECKOUT, so make sure to click on this button once all of the details have been entered and proceed with payment.

I hope I managed to assist and hope you have a wonderful day.

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ref:_00D30oeGz._5004Q1yoYmP:ref

------Original Message ------From: Armstrong Ighodalo Omoregie [[email protected]] Sent: 2/27/2020 4:13 AM To: [email protected] Subject: Permission to use article in my PhD thesis

Hello,

I am a final year PhD student at Swinburne University of Technology Sarawak Campus and I will be submitting my thesis for examination in March 2020.

The below articles is part of my doctoral study and they have been published in Elsevier Journals;

Omoregie, A.I., Palombo, E.A., Ong, D.E.L., Nissom, P.M., 2020. A feasible scale-up production of Sporosarcina pasteurii using custom-built stirred tank reactor for in-situ soil biocementation. Biocatal. Agric. Biotechnol. 24, 101544. https://doi.org/https://doi.org/10.1016/j.bcab.2020.101544

Hence, I am seeking permission to include this paper as a chapter in my thesis.

Kind Regards,

ARMSTRONG I. OMOREGIE PhD Candidate (Industrial Biotechnology)

Centre for Sustainable Technologies

[NOTICE] This e-mail and any attachments are confidential and intended only for the use of the addressee. They may contain information that is privileged or protected by copyright. If you are not the intended recipient, any dissemination, distribution, printing, copying or use is strictly prohibited. The University does not warrant that this e-mail and any attachments are secure and there is also a risk that it may be corrupted in transmission. It is your responsibility to check any attachments for viruses or defects before opening them. If you have received this transmission in error, please contact us on +6082 255000 and delete it immediately from your system. We do not accept liability in connection with computer virus, data corruption, delay, interruption, unauthorised access or unauthorised amendment.

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Appendix B

Authorship indication forms

242

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3HUFHQWDJHRIFRQWULEXWLRQ 'DWHBBBBBBBB Brief GHVFULSWLRQRI\RXUFRQWULEXWLRQWRWKHµSDSHU¶ ([WHUQDOVXSHUYLVRUSURYLGHGVRPHIXQGLQJIRUWKHSURMHFWDQGUHILQHGWKHPDQXVFULSW Fourth Author

Name: Assoc. Prof Peter Morin Nissom Signature:______

Percentage of contribution: 10% Date: _11 _ / _ 11 _ / _2019 _ _ _

Brief description of your contribution to the ‘paper’: Coordinating supervisor, co-designed the methodology, provided funding support for the project and revised manuscript.

Principal Coordinating Supervisor:

Name: Assoc. Prof Peter Morin Nissom Signature:______

Date: _1 1_ / _11 _ / _2019 _ _ _

In the case of more than four authors please attach another sheet with the names, signatures and contribution of the authors.

Authorship Indication Form Swinburne Research

Authorship Indication Form

For PhD by Publication candidates

NOTE This Authorship Indication form is a statement detailing the percentage of the contribution of each author in each published ‘paper’. This form must be signed by each co-author and the Principal Coordinating Supervisor. This form must be added to the publication of your final thesis as an appendix. Please fill out a separate form for each published paper to be included in your thesis.

DECLARATION We hereby declare our contribution to the publication of the ‘paper’ entitled: Utilization of custom-built reactor to scale-up the production of Sporosarcina pasteurii from small-scale to large-scale for in-situ soil biocementation

First Author

Name: Armstrong Ighodalo Omoregie Signature: ______

Percentage of contribution: 85% Date: 07/11/2019

Brief description of contribution to the ‘paper’ and your central responsibilities/role on project: I was the primary author of the article which contained contents arising from my PhD study. My tasks in this paper consisted of co-desigining the methodology, conducting literature review, performing laboratory experiments, interpreting and analyzing the results, writing the manuscript and corresponding author.

Second Author

Name: Prof Enzo A. Palombo Signature:______

Percentage of contribution: 3%

Brief description of your contribution to the ‘paper’: Date: _09 _ / _11 _ / _2019 _ _ _ Associate supervisor, provided feedback and correction to the manuscript.

Third Author

Name: Dr. Dominic Ek Leong Ong Signature: ______

Percentage of contribution: 5% Date: _08 _ / _11 _ / _2019 _ _ _

Brief description of your contribution to the ‘paper’: External supervisor, co-desianged some of the methodology, provided funding for the research and orrection to the manuscript. Fourth Author

Name: Assoc. Prof Peter Morin Nissom Signature:______

Percentage of contribution: 7% Date: _11 _ / 11_ _ / 2019_ _ _ _

Brief description of your contribution to the ‘paper’: Coordinating supervisor, co-designed the methodology, provided funding support for the project and feedback for the manuscript.

Principal Coordinating Supervisor:

Name: Assoc. Prof Peter Morin Nissom Signature:______

Date: _11 _ / 11_ _ / 2019_ _ _ _

In the case of more than four authors please attach another sheet with the names, signatures and contribution of the authors.

Authorship Indication Form Swinburne Research

Authorship Indication Form

For PhD by Publication candidates

DECLARATION We hereby declare our contribution to the publication of the ‘paper’ entitled: Biocementation of sand by Sporosarcina pasteurii strain and technical-grade cementation reagents through surface percolation treatment method

First Author

Name: Armstrong Ighodalo Omoregie Signature: ______

Percentage of contribution: 85% Date: 07/11/2019

Second Author

Name: Prof Enzo A. Palombo Signature:______

Percentage of contribution: 4%

Brief description of your contribution to the ‘paper’: Date: _09 _ / _11 _ / _2019 _ _ _ Associate supervisor, provided feedback and correction to the manuscript.

Third Author

Name: Dr. Dominic Ek Leong Ong Signature: ______

Percentage of contribution: 3% Date: _8 _ / _11 _ / _ 2019_ _ _

Brief description of your contribution to the ‘paper’: External supervisor and supported in providing correction to the manuscript. Fourth Author

Name: Assoc. Prof Peter Morin Nissom Signature:______

Percentage of contribution: 10% Date: _11 _ / _11 _ / _2019 _ _ _

Brief description of your contribution to the ‘paper’: Coordinating supervisor, co-designed the methodology, provided funding support for the project and feedback for the manuscript.

Principal Coordinating Supervisor:

Name: Assoc. Prof Peter Morin Nissom Signature:______

Date: _11 _ / _11 _ / _2019 _ _ _

In the case of more than four authors please attach another sheet with the names, signatures and contribution of the authors.

Authorship Indication Form