Use of Plant Cell Cultures in Biocatalyst Development for Bio-Desulfurization

Use of Plant Cell Cultures in Biocatalyst Development for Bio-desulfurization

A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Sciences and Engineering

2019

Maha Mohammed Al-Harbi

School of Chemical Engineering and Analytical Sciences

Contents List of figures ...... 5 List of Tables ...... 10 List of Abbreviations ...... 11 Nomenclature ...... 12 Abstract ...... 13 Declaration ...... 14 Copyright ...... 15 Dedication ...... 16 Acknowledgments ...... 17 Chapter 1 Introduction ...... 18 1.1. Background ...... 18 1.2. Aims and objectives ...... 20 1.3. Scope ...... 22 1.4. Thesis organization ...... 22 Chapter 2 Literature Review ...... 23 2.1. World Energy Consumption...... 23 2.2. Sulfur Dioxide (SO2) ...... 24 2.3. Global Anthropogenic Consequences of Sulfur Dioxide ...... 25 2.4. Sulfur Compounds in Crude Oil ...... 26 2.4.1. Crude Oil Fractions ...... 27 2.5. Sulfur Content Regulations ...... 29 2.5.1. Approach for Limiting SO2 Emissions ...... 30 2.6. Dibenzothiophenes (DBT) ...... 31 2.7. Methods of Desulfurization ...... 33 2.7.1. Desulfurization by Ionic Liquids (ILs)...... 33 2.7.2. Hydrodesulfurization ...... 33 2.7.3. Oxidative Desulfurization ...... 34 2.7.4. Physical Adsorption ...... 34 2.7.5. Biodesulfurization ...... 35 2.8. Biodesulfurization Pathways ...... 35 2.8.1. Kodoma Pathway ...... 36 2.8.2. The 4S Pathway ...... 37 2.8.3. Angular Deoxygenation Through Oxidative C-C Bond Cleavage and DBT Mineralization..43 2.8.4. Anaerobic Biodesulfurization Via Reductive C-S Bond Cleavage ...... 45 2.9. BDS and its Lack of Commercial Application in Industry ...... 46 Chapter 3 Plant Cell Culture in Biotechnology ...... 48 3.1. Plant Cell Culturing Protocols in Vitro ...... 48 3.2. Plant Cell Culture Applications ...... 50 3.3. Plant Species Used in this Work ...... 51 3.3.1. Arabidopsis thaliana ...... 52 3.3.2. Armoracia rusticana (horseradish) ...... 53 3.3.2. Nicotiana tabacum (tobacco) ...... 53 3.4. Common Enzymatic Approach in the different Species of Organisms (human, animal, plant and microorganism) which can Oxidise Sulphur Components ...... 54

2 3.5. The Effect of Oil Contacting Plants ...... 55 3.6. Hairy Roots: Mechanism of Transformation ...... 56 Chapter 4 Experimental Materials and Methodology ...... 59 4.1. Molecular Docking Procedure ...... 60 4.2. Medium Preparation ...... 62 4.2.1. Preparation of Essential Compounds for Medium ...... 66 4.2.2. Preparation of Essential Components for Chemical Analyses ...... 66 4.2.3. Preparing Saline Solution ...... 67 4.2.4. Preparation of pH Adjustment Chemicals ...... 67 4.3. Plants, Callus, Cell Suspension and Hairy Root Cultures ...... 67 4.3.1. Initiation of Cell Suspension Cultures Starting from the Seeds ...... 67 4.3.2. Initiation of Suspension Cells from the Explants Grown in Soil ...... 69 4.3.3. Cell Culture Sieving and Maintenance ...... 70 4.3.4. Contamination Test ...... 70 4.4. Experimental Preparation Procedures ...... 71 4.4.1 Incubation of Nicotiana tabacum Cells into Different Growth Media ...... 74 4.5 Analytical Techniques ...... 75 4.5.1. Fresh and Dry Weight Measurements ...... 75 4.5.2. Sample Preparation for GC-FID and HPLC Analysis ...... 77 4.5.3. Sugar Analysis by High Performance Liquid Chromatography (HPLC)...... 79 4.5.4. DBT and 2HBP Analysis ...... 81 4.6. Cell Viability Tests ...... 92 4.6.1 Removing Crude Oil by Washing with Saline Technique ...... 92 4.6.2 Cell Viability Test by Using Fluorescein Diacetate ...... 93 4.6.3. Monitoring Nicotiana tabacum Culture Growth Subsequent to Contacting with Crude Oil at Various Length of Times ...... 95 4.7. Hairy Roots ...... 96 Chapter 5 Theoretical Background for the Kinetic Modelling ...... 100 5.1 Some Fundamental Concepts ...... 102 5.2 Michaelis-Menten Kinetics for Enzymatic Reactions...... 104 5.3 Monod Kinetics for Growth ...... 105 5.4 Maintenance Energy Concept ...... 106 5.5 Substrate Uptake and Yield Factors (Yields) ...... 106 5.6 A Kinetic Model for Batch Cultures of Nicotiana tabacum Cell Suspensions ...... 108 5.7 Summary...... 111 Chapter 6 Results and Discussion: Initiation of Various Plant Cell and Hairy Root Cultures ...... 112 6.1. Results of Molecular Docking for Biodesulfurization Enzymes ...... 114 6.2 Metabolic Reactions for Biodesulfurization ...... 118 6.3 Results of Plant Cell Suspension Culture Initiation and Maintenance...... 120 6.4 Initiation of Horseradish and Nicotiana tabacum Hairy Roots ...... 128 6.5 Summary...... 136 Chapter 7 Results and Discussion: Use of (Nicotiana tabacum) Cell Suspension Cultures for Biodesulfurization ...... 137 7.1 Nicotiana tabacum Growth and Biodesulfurization in Aqueous Media ...... 140 7.1.1 Effect of Air Exchange on Nicotiana tabacum Cell Culture in MS and SFM with 100ppm DBT ...... 140 7.1.2. Effect of DBT Concentration on Nicotiana tabacum Growth in MS Aqueous Medium. ..148

3 7.1.3 Six-hourly Sampling of Nicotiana tabacum Cultures in MS Aqueous Medium Supplemented with 200ppm DBT and in SFM Aqueous Medium Supplemented with 100ppm DBT ...... 152 7.1.4 Nicotiana tabacum Kinetic Model Applications for Cultures in Aqueous Media ...... 156 7.2 Nicotiana tabacum Growth and Biodesulfurization in Aqueous Medium with Addition of Crude Oil ...... 164 7.2.1 FDA Test for the Effect of Crude Oil on Cell Viability ...... 164 7.2.2 Subsequent Cell Growth in MS Medium for 2 weeks After Contact with Crude Oil for Various Lengths of Time ...... 167 7.2.3 Biodesulfurisation Using MS and Sulfur-free MS Media and Crude Oil ...... 170 7.2.4 Effect of Sulfur Compounds Naturally Occurring in the Crude Oil on Cultures in Sulfur- Free-Medium ...... 178 7.2.7 (Nicotiana tabacum) Kinetic Model Application for the Biodesulfurization Experiment in Presence of Crude Oil ...... 181 Chapter 8 Conclusion and Recommendations ...... 184 References ...... 190 Appendix 1 Example of MATLAB Code of Kinetic Model ...... 198

4 List of figures Figure 2-1 Cooperation and Development members (OECD) and non-members (non-OECD) petroleum and other liquid fuels consumption. Source: Outlook (2016)...... 24

Figure 2-2 Sectorial trends in global, Chinese, and Indian SO2 emissions from 1990 to 2010, Teragram

SO2. Note different scales: i.e., India about 1/3 of China and the latter 1/3 of the world emissions (Klimont and Z Smith, 2013)...... 25 Figure 2-3shows acidic and non-acidic organic sulfur compounds in crude oil (Esmail Alkhalili et al., 2017)...... 26 Figure 2-4shows structures of the refractory sulfur compounds that are present in the petroleum streams (Kulkarni and Afonso, 2010)...... 27 Figure 2-5The maximum sulfur limit in crude oil across the globe (Association, 2009)...... 29 Figure 2-6 Direct mechanism for the hydrodesulfurization of dibenzothiophene (Mudt et al, 2006)...... 34 The Figure 2-7 Kodama pathway of DBT degradation (Srivastava, 2012) ...... 37 Figure 2-8 The 4S pathway of DBT biodesulfurization (Bhanjadeo et al., 2018)...... 43 Figure 2-9 Overview of the bacterial degradative pathway for DBT via angular dioxygenation. The structures shown in brackets have not been characterized. The arrows, with solid and broken lines indicate the enzymatic and spontaneous conversion, respectively (Hideaki Nojiri, 2001)...... 44 Figure 2-10 Principle of reductive DBT desulfurization pathway by Desulfovibrio desulfuricans M6 , adapted from(Kim et al., 1990)...... 45 Figure 3-1 Agrobacterium rhizogenes infection mechanism (Golikov)...... 57 Figure 4-1Cartoon representation of the X-ray structure of (a) NADP (H) Reductase (PDB ID: 4hfm), (b) Alfalfa feruoyl coenzyme A 3-O-methyltransferase (PDB ID: 1sus) and (c) AtDHNAT1, a 1, 4- dihydroxy-2-naphthoyl-CoA thioesterase (PDB ID: 4k02). In all three figures Helix, Beta sheet, and Loop. (PDB = Protein Data Bank)...... 61 Figure 4-2 Metal sieves where the cells should be poured and use the glass bar to press the cells gently to the baker...... 70 Figure 4-3 Flasks stoppered with cotton and aluminium foil and for crude oil experiments replace the cotton with rubber bung and wrap it with Para-film...... 71 Figure 4-4 5ml sterilized measuring cylinder ...... 72 Figure 4-5The picture shows 2ml Nicotiana tabacum cells and 2ml crude oil in 20ml of medium in airtight bottles for the batch experiments...... 73 Figure 4-6The picture Shows 2 ml Nicotiana tabacum cells in 20 ml of MS medium in Duran bottles...... 73 Figure 4-7 The vacuum filter to separate the cells from the medium in the culture samples...... 75 Figure 4-8 Fresh and dry cells on Whatman filter papers...... 76 Figure 4-9 A 22mm diameter and 0.45 µm cellulose nitrate membrane syringe filter...... 77 Figure 4-10 Crude oil was mixed with the medium; it was difficult to inject the crude oil and the medium separately in the small vial...... 78 Figure 4-11 The crude oil and the medium were separated by using a centrifuge 10000rpm for 10 minutes...... 78 Figure 4-12The crude oil and the medium in separate vial ready for GC-FID and HPLC analysis...... 78 Figure 4-13 Calibration curve for sugar analysis...... 80

5 Figure 4-14 HLPC chromatogram of sugar (sucrose, glucose, and fructose) mixture (area against time), retention time of sucrose, glucose, and fructose were 11 minutes; 12.968 minutes; and 15.803 minutes respectively...... 80 Figure 4-15 Blue colour with Gibbs’ reagent indicating the presence of 2-HBP at different concentration of the standard...... 82 Figure 4-16 The 2-HBP standards at zero minutes on the left and after half an hour on the right...... 82 Figure 4-17Calibration Curve for 2-HBP...... 82 Figure 4-18 Calibration curve of DBT dissolved in absolute ethanol in the left, and 2-HBP dissolved in absolute ethanol in the right...... 84 Figure 4-19 Calibration curve of DBT dissolve in medium in the left, and 2-HBP dissolve in medium in the right...... 84 Figure 4-20 Calibration curve of DBT dissolve in distilled water in the left, and 2-HBP dissolve in distilled water in the right...... 85 Figure 4-21 Calibration curve from running the standards in HP-1, Methyl Siloxane column, DBT dissolve in medium in the left and 2-HBP dissolve in medium in the right...... 85 Figure 4-22 Ethanol peak at retention time 1.1 minute in pure ethanol and ethanol in fresh medium elute from the DB-5MS...... 86 Figure 4-23 Calibration curves for 200ppm of DBT standards which were prepared in different solution in the DB-5MS column (area against time). A- Shows ethanol peaks at 1.1, B-Shows DBT peaks at 17 min in fresh , ethanol and no peak in distilled water at 17 min ...... 87 Figure 4-24 Calibration curves for 200ppm of 2-HBP standards which were prepared in different solution in the DB-5MS column (area against time) A-Shows ethanol peaks at 1.1, B-Shows 2-HBP peaks at around 14 min in fresh medium, ethanol and distilled water...... 88 Figure 4-25 Some Sulfur compounds area in pure Crude oil...... 89 Figure 4-26 200ppm standards for DBT peak at 1.5 and 2-HBP peak at 1.9 at 100:1 ratio (area against time)...... 90 Figure 4-27 The pure crude oil peaks in the HP-1 column (area against time)...... 91 Figure 4-28 Reaction scheme of fluorescein diacetate hydrolysis catalyzed by plant esterases (PE) (Vitecek, 2007 et al.,) ...... 93 Figure 4-29 A- Fresh and dried cells after contacting free medium crude oil, which show the sticking of crude oil on the cells. B- Fresh and dried cells after contacting crude oil with medium which show less crude oil sticking on the cells...... 94 Figure 4-30 a Single-Vial of Agrobacterium rhizogenes from ATCC...... 98 Figure 4-31Agrobacterium rhizogenes growth in 2 different media right YEB left ATCC medium...... 98 Figure 4-32 Contamination in Agrobacterium growth in the revival step before transfer the cells to the suspension culture of the bacteria for inoculation...... 99 Figure 4-33 Suspension culture of Agrobacterium growth in 2 different media YEB and ATCC medium to prepare the bacteria inoculation...... 99 Figure 4-34. Infecting the leaves by A. Making a wound. B. Using fine needle to inject the bacteria suspension culture into the leaves C. Using fine needle to inject the bacteria suspension culture into the roots...... 99 Figure 5-1 Hydrolysis of sucrose into glucose and fructose by the invertase enzyme...... 104 Figure 5-2 Lineweaver-Burk plot...... 105 Figure 6-1 Experimental strategy for investigating the biodesulfurization potential of plant cell culture (Stage 1) ...... 113

6 Figure 6-2 (a) Binding of DBT molecule to the binding site of NADP (H) Reductase, and (b) Binding of HBPS to the binding site of Alfalfa feruoyl coenzyme A 3-O-methyltransferase. Where, a1 and b1 is Cartoon representation, while a2 and b2 is hydrophobic surface representation...... 114 Figure 6-3 Graphical presentation of the theoretical affinity constants of 4hfm = NADP(H) Reductase, 1sus = Alfalfa feruoyl coenzyme A 3-O-methyltransferase, and 4k02 =AtDHNAT1, a 1,4-dihydroxy-2- naphthoyl-CoA thioesterase, HRP = Horseradish peroxidase, DszC= Dibenzothiophene monooxygenase, DszA = Dibenzothiophene sulfone monooxygenase, DszB =2'-hydroxybiphenyl-2- sulfinate desulfinase, toward DBT, DBTO, DBTO2, and HBPS ligands using the data presented in Table 6-1and 6-2...... 117 Figure 6-4 The proposal of 4S for biodesulfurization pathway in plant cells. Where 1sus Nicotiana tabacum enzyme, 4k02 in Arabidopsis thaliana, HRP in Horseradish and 4hfm in all plants and it is the enzyme which is equivalent to NADP (H) and DszC in bacteria, DszB specific for last step to convert HBPS to HBP...... 118 Figure 6-5 Metabolic reactions for biodesulfurization that show stoichiometric and redox balances...... 120 Figure 6-6 The initiation of Arabidopsis thaliana cell suspension culture starting from the seeds. ...122 Figure 6-7 The initiation of Nicotiana tabacum cell suspension culture starting from the seeds...... 123 Figure 6-8 Armoracia rusticana (Horse Radish) growing in compost in a pot...... 123 Figure 6-9 The initiation of Horseradish cell suspension culture starting from the leaves collected from the whole plant...... 124 Figure 6-10 The plant leaves starting to friable to create the callus, Nicotiana tabacum seems to be more active and faster than Horseradish...... 125 Figure 6-11 In different stages of Nicotiana tabacum callus initiation and maintenance...... 125 Figure 6-12 Nicotiana tabacum callus and suspension cultures easily get contaminated ...... 126 Figure 6-13 Nicotiana tabacum plant cells aggregates before sieving...... 127 Figure 6-14 Nicotiana tabacum fine cell aggregates after sieving...... 127 Figure 6-15Heavy Contamination for (Exp2)...... 133 Figure 6-16 The leaves lost their green color and nearly died (Exp4)...... 133 Figure 6-17Non-heavy Contamination (Exp5)...... 133 Figure 6-18No hairy roots (Exp6)...... 134 Figure 6-19 Contamination and wilted the roots. (Exp7)...... 134 Figure 6-20 Positive results (the root start to grow after 10 days) right Horseradish, lift Nicotiana tabacum (Exp8)...... 134 Figure 6-21 The growth was successful of the roots in Nicotiana tabacum leaves which appear after a week and then continue grow , ready to separate from the leaves...... 135 Figure 6-22 The separated hairy roots from the leaves in the solid MS free hormone medium to liquid MS free hormone medium...... 135 Figure 7-1 Experimental strategy for investigating the biodesulfurization potential of Nicotiana tobacum cell culture (Stage 2) ...... 138 Figure 7-2 Nicotiana tabacum batch culture control experiment in MS medium in flasks with cotton bung allowing air exchange (Code.MS). A- The experiment run with one sample each time, ...... 142 Figure 7-3 Nicotiana tabacum batch culture in MS medium in Duran bottles with no air exchange (Code.MS.D)...... 143 Figure 7-4 Nicotiana tabacum batch culture in SFM medium with 100ppm DBT in flasks with cotton bung allowing air exchange (Code.SF.100). A- The experiment run with one sample each time, .....146

7 Figure 7-5 Nicotiana tabacum batch culture in SFM medium with 100 ppm DBT in Duran bottles with no air exchange. This experiment was run in duplicate (Code.SF.100.D)...... 147 Figure 7-6 The production of 2-HBP during the culture of Nicotiana tabacum in SFM.D.100ppm DBT and SFM.100ppmDBT(A)...... 147 Figure 7-7 Nicotiana tabacum batch culture in MS medium supplemented with 100ppm DBT in flasks with cotton bung allowing air exchange (Code.MS.100). A- The experiment run with one sample each time, B- The average results of two other experiments with same conditions as A...... 150 Figure 7-8 Nicotiana tabacum batch culture in MS medium supplemented with 200ppm DBT in flasks with cotton bung allowing air exchange (Code.SF.200). A- The experiment run with one sample each time, B- The average results of two other experiments with same conditions as A...... 151 Figure 7-9 The production of 2-HBP during the culture of Nicotiana tabacum in MS.100ppm DBT (A) and MS.200ppmDBT(A)...... 152 Figure 7-10 Nicotiana tabacum batch culture (dry weight) g L-1, sugar and DBT analysis. The samples were collecting every 6 hours for 90 hours’ batch, in MS medium supplemented with 200ppm DBT (Code.MS.200/6h)...... 154 Figure 7-11 Nicotiana tabacum batch culture (dry weight) g L-1, sugar and DBT analysis. The samples were collecting every 6 hours for 90 hours’ batch, SFM medium supplemented with 100ppm in air exchange flasks (Code.SF.100/6h)...... 154 Figure 7-12 The production of 2-HBP during the culture of Nicotiana tabacum in SFM.100ppm DBT and MS.200ppm DBT every 6 hours (This experiment was not run in duplicate) ...... 155 Figure 7-13 The relationship between fresh and dry weight (g L-1) in MS 200ppm DBT and SFM 100ppm DBT in every 6 hrs collection time...... 155 Figure 7-14 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain Vmaxand⁡ KM in MS normal medium in air exchange condition...... 158

Figure 7-15 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax'1 in MS normal medium in air exchange condition...... 158

Figure 7-16 Semi-log plot of phase 2 for determination of the maximum specific growth rate μmax'2 in MS normal medium in air exchange condition...... 159 Figure 7-17 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain Vmaxand⁡ KM in MS normal medium with 100ppm DBT in shake flasks in air exchange...... 159

Figure 7-18 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax’1 in MS normal medium in air exchange condition supplemented with 100ppm DBT...... 160

Figure 7-19 Semi-log plot of phase 2 for determination of the maximum specific growth rate μmax'2 in MS normal medium in air exchange condition supplemented with 100ppm DBT...... 160 Figure 7-20 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain Vmaxand⁡ KMin SFM medium in air exchange condition supplemented with 100ppm DBT...... 161

Figure 7-21 Semi-log plot of one phase for determination of the maximum specific growth rate μmax' in SFM medium in air exchange condition supplemented with 100ppm DBT...... 161 Figure 7-22 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in MS normal medium with air exchange condition. (Code.MS)...... 162 Figure 7-23 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in MS normal medium supplemented with 100ppm of DBT with air exchange condition. (Code.MS.100) .162 Figure 7-24 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in SFM medium supplemented with 100ppm of DBT with air exchange condition.(Code.SF.100) ...... 163

8 Figure 7-25 Nicotiana tabacum culture and sugar up take ,the cells was growing MS normal medium after contacting crude oil in different length of time every two hours (2,4,6,8, 24) h contacting time after wash them by using saline solution...... 169 Figure 7-26The normal dry cells in crude oil free MS normal medium in the left and in the right the last sample at 408 hrs shows wilted dry cells in MS normal medium after washing from 2hrs contacting time with crude oil...... 170 Figure 7-27 Nicotiana tabacum batch culture in MS normal medium (dry weight) g L-1 and sugar analysis, the growth was in Duran bottle (No air exchange) presence of crude oil and 100ppm DBT (Code.MS.100.D.O)...... 172 Figure 7-28Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis, the growth was in Duran bottle (No air exchange) presence of crude oil and 100ppm DBT (Code.SF.100.D.O)...... 172 Figure 7-29 The production of 2-HBP during the culture of Nicotiana tabacum in MS and SFM medium with 100ppm DBT in presence of crude oil...... 173 Figure 7-30 Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis and 200ppm DBT, the growth was in Duran bottle (No air exchange) presence of crude oil (Code.SF.200.D.O)...... 175 Figure 7-31Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis, the growth was in Duran bottle (No air exchange) presence of crude oil with 300ppm DBT (Code.SF.300.D.O)...... 176 Figure 7-32Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis and 400ppm DBT, the growth was in Duran bottle (No air exchange) presence of crude oil (Code.SF.400.D.O)...... 176 Figure 7-33 The production of 2-HBP during the culture of Nicotiana tabacum in SFM supplemented with 100, 200, 300 and 400 ppm DBT in the presence of crude oil...... 177 Figure 7-34 2-HBP concentration in crude oil samples from cultures of Nicotiana tabacum in SFM medium in air tight condition with different concentrations of DBT addition...... 179 Figure 7-35 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain Vmaxand⁡ KM in SFM medium in no air exchange condition supplemented with 100ppmDBT in presence of crude oil...... 182

Figure 7-36 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax'1 in SFM medium in air exchange condition supplemented with 100ppm DBT in presence of crude oil. 182

Figure 7-37 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax'2 in SFM medium in air exchange condition supplemented with 100ppm DBT in presence of crude oil. 183 Figure 7-38 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in SFM medium supplemented with 100ppm of DBT with air tight condition in presence of crude oil (Code.SF.100.D.O)...... 183

9 List of Tables Table 2-1Sulfur contents of various crude oils at the surface conditions (Wauquier, 1995)...... 28 Table 2-2 Summarizes the prevailing world-class emission standards (with permission ICCT) (Kodjak, 2015)...... 31 Table 2-3 Some examples of microorganisms which can desulfurize the DBT in the 4S pathway...... 40 Table 2-4 Shows the biodesulfrization capabilities of various microorganisms optimized conditions ..41 Table 2-5 Biodesulfrization activity by some bacteria strains and the percentage of sulfur removed from Crude oil fractions and their sulfur contents (Mohebali and Ball, 2008)...... 42 Table 2-6 Desulfurization of Petroleum by Different bacteria strain (Esmail Alkhalili et al., 2017). ....42 Table 3-1:The plants taxonomy of the plant which were using in this study...... 52 Table 3-2 How oil coming into contact with whole plants affects the rate of respiration and transpiration (Setti et al., 1997, El-Gendy and Nassar, 2018) ...... 56 Table 4-1: The difference between normal and sulfur free Murashige and Skoog basal medium composition...... 63 Table 4-2 Supplements for the Murashige and Skoog (MS) media. Sulfur free MS (SFM) additionally was supplemented with 100 – 400ppm DBT...... 64 Table 4-3 Batch experiments operated with Nicotiana tabacum cells cultures in different types of media and different conditions. These media codes* are used later in Chapter 7 ...... 74 Table 4-4 Yeast extract broth components (YEB) and (www.atcc.org)...... 98 Table 6-2 Summary of Molecular Docking Results in micoorganism (Rhodococcus erythropolis ) ...... 116 Table 6-1 Sammary of Molecular Docking Results in plant cells...... 116 Table 6-3The table is shown different methods of infection and conditions have been attempted to obtain the optimal conditions for hairy roots initiation ...... 131 Table 6-4 Continue the table is shown different methods of infection and conditions have been attempted to obtain the optimal conditions for hairy roots initiation ...... 132 Table 7-1 The table shows different experimental conditions with their codes summarised with their associated subsection and figure numbers...... 139 Table 7-2 Stoichiometric and kinetic parameters relating to (Nicotiana tabacum) growth and Sucrose and DBT consumptions in MS normal medium and SFM medium ...... 157 Table 7-4 Effect of crude oil on cell viability when its contact with mixture of MS medium in presence of crude oil for various lengths of time...... 165 Table 7-5 Effect of crude oil on cell viability when its contact with crude oil without medium for various lengths of time. (0h,1h, 12 days)...... 166 Table7-7 Stoichiometric and kinetic parameters relating to Nicotiana tabacum growth and Sucrose and DBT consumptions in SFM medium supplemented with 100ppmDBT in presence of crude oil .181

10 List of Abbreviations

BDS =Biodesulfrization

DBT =Dibenzothiophene

DBTO = DBT-5-oxlde=sulfoxide

DBTO2=DBT-S, S-dioxide? =sulfone

HPBS =2'Hydroxyblphenyl-2-sulflnate

2-HPB = 2-Hydroxybiphenyl

MS= Murashige and Skoog

SFM=Sulfur free Murashige and Skoog

GB5= Gamborg’s B5 medium

YEB =Yeast extract broth

ATCC= American Type Culture Collection

4MDBT=Miethyldibenzothiophen

4,6-DEDBT = 4,6-Diethyldibenzothiophen

API =American Petroleum Institute

US EPA =United States Environmental Protection Agency

GC-FID= Gas chromatography equipped with a flame ionization detector

HPLC= High Performance Liquid Chromatography

FDA= fluorescein diacetate rpm = revolutions per minute

PDB = Protein Data Bank

11 Nomenclature

F fructose concentration (gL-1) G glucose concentration (gL-1) -1 KF Monod constant for fructose uptake (gL ) -1 KG Monod constant for glucose uptake (gL ) -1 Kig glucose inhibition constant of fructose uptake rate (g glucose (L) ) -1 KS Michaelis-Menten constant for sucrose hydrolysis (gL ) -1 kd First order death rate kinetics constant (h ) -1 -1 rF rate of fructose formation from sucrose hydrolysis (gL h ) -1 -1 rFx rate of fructose consumption for growth (gL h ) -1 -1 rG rate of glucose formation from sucrose hydrolysis (gL h ) -1 -1 rGx rate of glucose consumption for growth (gL h ) -1 -1 rs rate of sucrose hydrolysis (gL h ) -1 -1 rx rate of biomass (dry weight) growth (gL h ) ∆S Percentage of sucrose concentration (gL-1) -1 -1 Vmax maximum rate of sucrose hydrolysis (gL h ) -1 xv viable biomass concentration (gL )

X0 inoculation cells dry weight g/l

X max inoculation cells dry weight g/l -1 YF/S stoichiometric yield of fructose from sucrose hydrolysis ((g fructose) (g sucrose) ) -1 YG/S stoichiometric yield of glucose from sucrose hydrolysis ((g glucose) (g sucrose) ) ’ -1 Y x/F yield of biomass formation from fructose ((g cells) (g fructose) ) ’ -1 Y x/G yield of biomass formation from glucose ((g cells) (g glucose) ) µ specific growth rate (h-1) -1 µmax maximum specific growth rate (h )

-1 휇푚푎푥⁡⁡⁡maximum specific growth rate of biomass in phase one (h ) ′ -1 휇푚푎푥⁡⁡⁡maximum specific growth rate of biomass in phase two (h )

Pavg Average Productivity (g cells l-1h-1) -1 -1 Pmax Maximum Productivity (g cells l h ) ms The maintenance constant (g Sucrose g cells-1 h-1)

12 Abstract

An impurity such as sulfur reduces the quality of fossil fuels and contributes significantly to air pollution, which has serious harmful effects on the environment. Therefore, this research project focuses on the development of biocatalysts based on plant cell cultures for the desulfurization of crude oil. Microbial biodesulfurization has been investigated for a few decades now. Yet there is no process based on biodesulfurization. There are many reasons for this, the main ones involve the difficulty of separating cells from the crude oil and hence the problem of consumption of the hydrocarbons by the cells leading to the loss of value of the crude oil, and high temperatures of the crude oil not being suitable for microbial cultures. The biogenesis of sulfur compounds in fossil fuels is caused by the death and decomposition under high temperature and pressure in the earth’s crust of the large populations of plants and animals over geological time. Plants produce complex sulfur compounds and therefore must have enzymes that can degrade sulfur compounds. Use of plant cell cultures for biodesulfurization was the novelty of this project. One of the pathways of bacterial biodesulfurization was elucidated as the 4S pathway. Using the identified enzymes in the 4S pathway, equivalent enzymes in various plant species were searched using databases such as KEGG, and molecular docking software SwissDock. Specially for Nicotiana tabacum (tobacco) as the chosen plant for this research, feruoyl coenzyme A 3-O-methyltransferase (1sus) was 7.8 times more efficient than 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB) enzyme used in the last step in 4S pathway in a biodesulfurizing micoorganism like Rhodococcus erythropolis. Three plant species, Arabidopsis thaliana, Nicotiana tabacum (tobacco), Armoracia rusticana (horseradish) were cultured. Nicotiana tabacum was the chosen plant for the biodesulfurization experiments due to the good growth characteristics and minimal contamination problems. Dibenzothiophene (DBT) was the model sulfur compound used for testing the biodesulfurisation ability of the cultures. The control experiments and biodesulfurization experiments were performed in aqueous medium in shake flasks using Murashige-Skoog (MS) and sulfur-free Murashige-Skoog (SFM) media supplemented either 100 or 200ppm DBT. The MS with 100ppm DBT experiment had the highest maximum biomass concentration at 24.46 g/l by day 15, 95% of DBT was degraded by day 15 and the average concentration of 2-Hydroxyblphen (2-HBP) produced was 66.3ppm. In advance of adding crude oil to the biodesulfurization experiments some tests were done and confirmed that cells could stay alive after contacting with crude oil, this was achieved by looking the cells treated with fluorescein diacetate staining (FDA) under UV microscope. The cells were allowed to have contact with crude oil for 24 h then washed and grown in MS normal medium for around 2 weeks. For the biodesulfurization experiments involving crude oil, the MS with 100ppm DBT and SFM with DBT at 100, 200, 300 and 400ppm were performed in airtight vessels for safety reasons. The 100ppm DBT concentration in SFM had the highest maximum biomass concentration at 26.7 g/l in day 21 and 97.7% of DBT degradation, and fluctuated concentrations of 2-HBP with an average of 46.31ppm. A kinetic model was used for some of the batch experiments; although preliminary attempt prediction was promising, nevertheless the kinetic model needs further refinement with further experiments in order to obtain the model parameters more accurately.

13 Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the "Copyright") and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses.

Dedication

To my mother's soul who taught me the meaning of life. To my father the origin of my success. To my brothers and sisters, may God bless them for their endless Love, Support & Encouragement. To my best friends Dalal, Kawthar and Diena.

16 Acknowledgments

First and foremost, I would like to thank and praise and show my gratitude to Al-Mighty God, who blessed my life and supported my efforts, and without whose help I could never have achieved anything in life.

I wish to express my sincerest gratitude to my supervisor, Professor Ferda Mavituna, for her inspiring instruction and patient guidance during the study of my PhD, which has a significant impact not only on my academic study but also personal life.

I want to give my heartfelt thanks to people who have offered me generous help and made the completion of the whole thesis possible. my appreciation to the technical staff, Thomas Szpitter, Shahla Khan. Dr.Nasser Al-Shanti for providing UV microscope from Manchester Metropolitan University . as well as my friends Khasim Cali , Abdullatif Alfutimie , Abdulrazzaq Alshammari and Hassan Abdullahi who have made great efforts to help with various aspects of my development. Special thanks to my colleagues Hao Yu and Lixing Gu for sharing the literature and invaluable assistance during my PhD.

Finally, I thank my parents and my family for supporting me throughout all my studies at University, without them I would not have come this far.

17 Chapter 1 Introduction

1.1. Background In many countries, there are legislative regulations on the use of environmentally- friendly fuel for transportation. This includes the use of bio-based products such as biodiesels or additions of bioethanol to the conventional fuels with the aim of reducing negative environmental impacts.

Apart from reducing the contribution of fossil fuels combustion to the carbon footprint, there are also other major concerns. Obviously, fossil fuels contain some level of nitrogen oxides (NOx), sulfur, and other particulate matters that can pose major health risks. When sulfur is released to the atmosphere, it can cause many environmental and health problems like heart diseases, asthma, and respiratory illnesses (PETROLIUMSadare et al., 2017); Knudsen et al., 1999).

There are coordinated efforts among international communities aimed at reducing or solving these air pollution problems. Some of them include signing agreements to reduce, control, monitor, and prevent gaseous emissions. For instance, Canada, America, and

European countries signed agreements to reduce SO2 emissions in 1979. In 1993, the same countries introduced the Clean Air Act that banned sales and supply of diesel oil -1 with concentrations of SO2 of more than 500 mg kg (Soleimani et al., 2007). They also put the target to be at 50 mg kg-1 in 2005. Five years later, the European Union set its limit at 350 mg kg-1. (Knudsen et al., 1999).The United States and Canada agreed that the maximum permissible sulfur content of diesel will be 30 and 15 mg kg-1 respectively (Avidan A, 2001). The US was planning to push down the sulfur contents in diesel to 10 mg kg-1 by the year 2010 (Kilbane Ii, 2006).

Biodesulfurization is one of the commonly used methods to remove sulfur from crude oil. Despite many decades of research, microbial biodesulfurization still has not been applied at a large scale. The biogenesis of sulfur in fossil fuels is of plant and animal origin. Plants can produce many complex compounds that contain sulfur such as organosulfur compounds. Numerous crucial S-containing compounds are derived directly or indirectly from cysteine, methionine, glutathione and, phytochelatins. The hypothesis is therefore, if

18 the plants can synthesize a range of complex sulfur-containing compounds, then they should have an enzyme mechanism that can also degrade such complex sulfur compounds since metabolic reactions are normally reversible. It is therefore expected that biodesulfurization activity will be found in plants. Since this is still a relatively novel research topic, the number of plant species that have been researched for their biodesulfurization activity is still relatively limited.

In this work, the biocatalytic activity of plants such as Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Armoracia rusticana (Horseradish) to degrade dibenzothiophene (DBT) as a common model sulfur compound which exist in crude oil and used in biodesulfurization studies, will be investigated using the plant cell suspension cultures. When the healthy leaves were obtained and ready for callus initiation at the same time some leaves were used to establish hairy root culture by infect the leaves with Agrobacterium rhizogenes which causes hairy roots disease. This hairy root culture can be used as a self-immobilization and improve biodesulfurization process. In the beginning, a high quantity of the plant cells will be obtained by bulking up the plant cell suspension cultures. The process will start from explant or seeds germination and then callus. Then cell suspension cultures will be initiated and then sub-culturing will continue until large enough cells are obtained for batch experiments. The suspension (liquid) medium that is normally used for the culturing and growth of plant cells will also be supplemented with DBT. Later on, plant cells will be treated with crude oil. However, it will firstly be determined if they can survive. This will be examined by using fluorescein diacetate FDA test. Their biodesulfurization activity will then be investigated. Batch cultures of plant cell suspensions in aqueous medium will be cultivated in shake flasks and their growth and sucrose (which is converted to glucose and fructose) consumption will be measured. These data will serve as the control for biodesulfurization experiments. Different concentrations of DBT will also be added to the free crude oil aqueous medium or aqueous medium in the presence of crude oil and its degradation consumption by the plant cells will be investigated (by measuring reduction of DBT concentration and formation of any products from it). High Performance Liquid Chromatography (HPLC) will be used for the determination of the products from sucrose (glucose and fructose) consumption, while Gas Chromatography (GC) will be used to monitor the degradation of DBT.

19 Added to the use cell suspension cultures of Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Armoracia rusticana (Horseradish), a novel aspect has recently been initiated: the use of hairy roots. Although hairy roots can become contaminated very easily at the early stage (as they are very fine and week), once they have been established they become strong and start to grow faster than the cells .As indicated above, the idea of using hairy root cultures is novel with regards to the biodesulfurization process. Hairy roots were initiated by infecting the plant with Agrobacterium rhizogenes, which can be easier to handle; as they self-immobilize as tangled roots and act like packing in bioreactors (Mohamed et al., 2015). Hairy roots are also less demanding in terms of medium requirements; they can survive longer without medium and are less prone to contamination at large scale operations. It is expected that hairy root cultures would increase the efficiency of the biodesulfurization process. Although these approaches could be novel and successful, they are still two orders of magnitude less efficient than biodesulfurization rate achievable for commercial use in crude oils and refinery streams (Mohamed et al., 2015).

1.2. Aims and objectives The aim of the project was to investigate the biodesulfurization capability of plant cell suspension and hairy root cultures in order to develop biocatalysts for biodesulfurization of crude oil. In order to achieve this aim, the project was performed in two stages as explained below: Stage 1: Choice of plant species and culture type: a. Initiation of callus and cell suspension cultures from Arabidopsis thaliana (thale cress), Armoracia rusticana (horseradish) and Nicotiana tabacum (tobacco) in order to obtain a healthy, easily grown and quickly bulked-up cell lines to be used in biodesulfurization experiments. b. Initiation of hairy root cultures from Armoracia rusticana (horseradish) and Nicotiana tabacum (tobacco) in order to obtain plant cell cultures that can be packed in to column bioreactors which could then be used in repeated cycles of crude oil pass and medium pass for biodesulfurization and culture maintenance. c. In order to confirm the presence of inherent biodesulfurization capability of these three-plant species, molecular docking procedures were used for the identified

20 enzymes of the bacterial biodesulfurization pathway (4S in Rhodococcus erythropolis) in comparison with the equivalent enzymes in these plants.

Stage 2: Biocatalyst Development for Biodesulfurization As explained in Chapter 6, only the Nicotiana tabacum cell suspension cultures grew well with less problems of contamination so that they were chosen for the following objectives of Stage 2. a. Control experiments: These involved batch cultures of Nicotiana tabacum cell suspensions in shake flasks using Murashige-Skoog (MS) medium with sucrose and plant growth regulators. b. Addition of different concentrations of dibenzothiophene (DBT) as the model sulfur compound in biodesulfurization studies to MS medium. c. Addition of different concentrations of dibenzothiophene (DBT) to sulfur-free MS medium. d. Additional control experiments for the effect of air-tight vessels (shake flaks and Duran bottles) on cell activity in MS medium and sulfur-free MS medium with DBT. e. Addition of crude oil with different concentrations of dibenzothiophene (DBT) to MS and sulfur-free MS medium in air-tight vessels (for safety reasons).

In these experiments, cell dry weight, sucrose, glucose and fructose concentrations were measured. When DBT was added, its concentration as well as 2- hydroxybiphenyl (2-HBP) which is the end product of biodesulfurization was also measured. When crude oil was present in the experiments, samples of it were also analysed for the contents of DBT and 2-HBP. Cell viability was also checked after contacting with crude oil for different lengths of time by two methods: fluorescein diacetate staining for viable cells and growth of cells after washing the crude oil off and transferring the cells in to MS medium.

21 1.3. Scope This work includes experimental, analytical and theoretical aspects. With regards to examining the plant cell cultures ability for biodesulfrization, this project is novel. All experimental work was performed in shake flasks. Experiments involved aqueous Murashige-Skoog (MS) basal medium supplemented with sucrose, MS medium with the addition of various concentrations of dibenzothiophene (DBT) as the model sulfur compound, sulfur-free MS medium supplemented with DBT, and experiments involving mixtures of aqueous media and crude oil which had various concentrations of DBT. Since the use of plant cell cultures for this purpose was novel, the comparison was made with microbial culture biodesulfurization. In order to make quantitative comparisons, a simple kinetic model was applied to growth, sugar consumption and the uptake of DBT. Moreover, the hairy roots of the plants were initiated by infecting with Agrobacterium rhizogenes with an intention of using hairy roots as self-immobilization in bioreactors. Unfortunately, because of heavy and persistent microbial contamination of hairy root cultures, this objective was not achieved.

1.4. Thesis organization This first chapter presents a brief background of the study, clarification for this research and aims and objectives. The literature review, which is presented in Chapter 2, is split into two chapters. The chapter starts by a discussion the world energy consumption and the harm of Sulfur Dioxide emission in particular. It details how sulfur has presence in crude oil. It explains biodesulfrization methods and finally the biodesulfrization pathways. The second part of the literature review is chapter 3 which deals with plant cell cultures and all relevant information about using plant cell cultures in biotechnology and the enzymatic approach and finally using hairy roots in vitro which can be produced from the plant. The routine procedures and materials are described in the materials and methods in Chapter 4. Chapter 5 is the theoretical background for the kinetic and metabolic modelling is summarized. The results and discussions are presented in 2 main chapters. Chapter 6 starts from moleculer docking results and the initiation of three different plants and hairy roots initiation from the plant leaves by using Agrobacterium rhizogenes. Chapter 7 presents the growth and sugar and biodesulfrization results in aqueous medium in different conditions and the growth in the present of crude oil that is after showing cell viability test when the cell comes into contact crude oil. In Chapter 8 the conclusions and recommendations for future studies and the appendix.

22 Chapter 2 Literature Review

This project is at the interface of a few research topics: sulfur compounds in fossil fuels and its harmful impact in our life, biodesulfurization which has involved microbial systems so far, metabolism of sulfur compounds and pathways of biodesulfurization, plant biotechnology especially plant cell suspension, and hairy root cultures. Some of the literatures relevant to these aspects are summarized in this chapter.

2.1. World Energy Consumption Energy is an important part of human life. There are generally two classifications of energy sources: renewable source (such as solar, wind, and geothermal) and non- renewable sources (made from fossil fuels). Human beings have generally started to realize how much the use of these energy sources can affect the environment. Hence, researchers have concentrated a lot of their efforts towards finding ways of reducing the use of non-renewable sources of energy and replace them with renewable ones. This is despite the fact that the energy produced form renewable sources is not enough for world’s demand. Therefore, the use of non-renewable sources of energy can still be used but the emission control technologies can be developed to limit the harmful chemicals discharged from the fossil fuel, such as coal, crude oil, and natural gas, emissions.

The International Energy Outlook (IEO, 2016) reports that every year there is increase of liquid fuel consumption. The same report confirmed that the consumption has increased from 90 to 100 million barrels per day from 2012 to 2015. It is expected that as populations grow, so will economies of different countries. This has lead to predictions that the consumption of liquid fuels by 2040 will reach 121 million barrels per day. Error! Reference source not found. shows the fuel consumption reported by IEO (2016) f rom 1990 to 2040 with Organization for Economic Cooperation and Development members (OECD) and nonmembers (non-OECD) (Outlook, 2016). This study focuses only on sulfur components in crude oil. Generally, energy consumption is known as the main cause for air pollution caused by chemicals such as carbon monoxide (CO), sulfur dioxide (SO2), nitrogen dioxide (NO2), persistent organic pollutants (POP), certain metals, certain volatile organic compounds, and nitrogen trichloride. However, this study will focus on sulfur dioxide (SO2) emission (Sadare et al., 2017).

barrels/day Million

Years

Figure 2-1 Cooperation and Development members (OECD) and non-members (non-OECD) petroleum and other liquid fuels consumption. Source:The international Energy Outlook (2016).

2.2. Sulfur Dioxide (SO2) Air is a mixture of gases and small solid and liquid particles, some of which originate from natural sources, whilst others are a consequence of anthropogenic human activity.

Harmful substances, called air pollutants, may be solids, liquids or gases (such as SO2) at large enough concentrations to have a negative effect to humans, animals, and plants (Alias Masitah, 2007). Air quality monitoring has, therefore, become increasingly important for governments and other organizations and is carried out by measuring air pollution levels. SO2 is an air pollutant, which is released from burning fuels such as coal, oil and gas for energy generation. Other air pollutants include nitrogen oxides (NOx) and particulate matter, from man-made and natural sources such as volcanic eruptions and diesel (Ahmed, 1999)

SO2 is a colorless toxic gas with a pungent smell; it is about 2.5 times heavier than air.

Furthermore, SO2 can be produced in the lower atmosphere and can form a sulfate aerosol with a diameter of 2.5 µm. This aerosol is very harmful for humans because it can cause respiratory illnesses (World Bank, 1999; Atlas et al., 2001). Sulfur dioxide reacts with rain to form harmful compounds, such as sulfate particles and sulfuric acid. The acid formed can cause damage to humans, animals, and plants (Soleimani et al., 2007).

High SO2 concentrations can be identified by a distinct odour of burning matches. It has been shown to cause health complications related to the respiratory system such as

24 asthma. Long-term and serious health conditions may also be caused by SO2. Examples include heart disease, bronchitis, and cardiovascular diseases. Environmental effects of

SO2 include acidification (acid rain) and formation of particulate matter, which disrupt the environment and ecosystems (Li, 2007); (Ibrahim, 2017).

2.3. Global Anthropogenic Consequences of Sulfur Dioxide The major source of SO2 is human industrial activities, which include the burning of coal, oil and gas that contains sulfur. Grübler (1998) reviewed available literature and empirical evidence and concluded that economic development plays a huge role in driving sulfur depositions. As economic development increases, energy use also increases and so does the rise of SO2 emissions (Ismail, 2016).

Figure 2-2 Sectorial trends in global, Chinese, and Indian SO2 emissions from 1990 to 2010, Teragram SO2. Note different scales: i.e., India about 1/3 of China and the latter 1/3 of the world emissions (Klimont and Z Smith, 2013).

Figure 2-2 shows trends of SO2 emissions from 1990 to 2010, in China, India and the world. It shows that global emissions in 2010 have decreased to below that in 2000, by around 3%. Although there was an increase in emissions over the period between 2000 and 2010 in China, India, Eastern Europe, Caucasus, and Central Asia, this increase in emissions was smaller than reductions in Europe and North America. Therefore, the global emissions show a net decrease (Klimont and Z Smith, 2013, Roser, 2018).

25 2.4. Sulfur Compounds in Crude Oil It has been established that biodesulfrization (BDS) technology has a great potential for the removal of sulfur from crude oil. The model sulfur compound dibenzothiophene (DBT) used in this study can now be used to determine the different amounts of sulfur in crude oil fractions around the world.

Sulfur is present in crude oil in abundance and has the third highest elemental concentration after carbon and hydrogen (Soleimani et al., 2007). It is present in its two main forms – organic and inorganic compounds (Javadli & Klerk, 2012). Elemental sulfur is rarely found in the oil as it steadily reacts with the hydrocarbons present when heat is applied (Wauquier, 1995).

The organic sulfur compounds found in crude oil, tend to exist in two separate forms, heterocyclic and non-heterocyclic compounds with most of the research in open literature focusing mainly on the former (Mohebali & Ball, 2008). Organic sulfur compounds can be classified as either acidic sulfur compounds which include mercaptans or thiols or non- acidic sulfides such as disulfides or thiophenes (Duissenov, 2012, 2013). This is shown in Figure 2-3 below. In addition, in Figure 2-4 the structures of the refractory sulfur compounds that are present in the petroleum streams are shown.

Figure 2-3shows acidic and non-acidic organic sulfur compounds in crude oil (Esmail Alkhalili et al., 2017).

Figure 2-4shows structures of the refractory sulfur compounds that are present in the petroleum streams (Kulkarni and Afonso, 2010). The acidic mercaptans generally contain a thiol (SH) group that contributes 0.1 to 15% of the total mass of the compound (Ryabov, 2009). Other acidic organic compounds are chemically neutral sulfides which account for a large fraction of the total sulfur present in the crude oil; ranging from 50 – 80%. Disulfides on the other hand, account for much smaller sulfur content with ranges from 7 to 15% (Ryabov, 2009). Another organic compound found in the crude oil is the neutral five membered cyclic and temperature resistant thiophenes and its derivatives. Due to their structure, they do not dissolve in water and exhibit chemical properties similar to aromatic hydrocarbons (Duissenov, 2013). In the same fashion as aromatic compounds, thiophenes are recalcitrant, requiring deep desulfurization processes to fully remove their sulfur atom (Esmail Alkhalili et al., 2017).

Most of the inorganic sulfur present within the crude oil is found either suspended or dissolved in the oil (Soleimani et al., 2007). One of these major sulfur compounds that are found in the oil after processing methods such as hydrodesulfurization, catalytic cracking, thermal cracking, and thermal decomposition during distillation is hydrogen sulfide – H2S (Wauquier, 1995).

2.4.1. Crude Oil Fractions Crude oil accounts for around 39% of fossil fuel energy; which is the largest energy source. Around 4 billion tons of crude oil, in its different constituents, is consumed annually around the world (Factbook, 2012). American Petroleum Institute (API) gravity is a measure of density which is used to identify the type of crude oil, as well as the sulfur content (Roser, 2018). Crude oil with sulfur content under 0.5% in weight is known as sweet crude oil. As the amount of sulfur is so small, this form of crude oil requires little

27 purification to remove the sulfur. Therefore it has higher fuel content in its original form, and it is relatively expensive. In comparison, crude oil with sulfur content above 0.5% in weight is known as sour crude oil, which requires further desulfurization before it can be sold, and this type of crude oil will therefore be used in this study (Petroleum.com, 2015). A variety of crude oil types with their country of origin and sulfur content can be seen in Table 2-1below. As this study focuses on desulfurization, other components found in crude oil such as nitrogen are not included.

Table 2-1Sulfur contents of various crude oils at the surface conditions (Wauquier, 1995).

Crude Oil Name Country of Origin Weight % Sulfur

Arabian Light Saudi Arabia 1.80

Arjuna Indonesia 0.12

Bonny Light Nigeria 0.13

Boscan Venezuela 5.40

Bu Attifel Libya 0.10

Cyrus Iran 3.48

Ekofisk North Sea (Norway) 0.18

Hassi Messaoud Algeria 0.14

Kirkuk Iraq 1.95

Kuwait Kuwait 2.50

28 2.5. Sulfur Content Regulations

Figure 2-5The maximum sulfur limit in crude oil across the globe (Association, 2009). Table 2-1 shows the different sulfur contents in various crude oils. From the Table, it can be observed that Arjuna Crude Oil, which has a very low amount of sulfur, would be the most expensive one whereas the Boscan Crude Oil would be the least expensive because of its very high sulfur content. Furthermore,

Figure 2-5, shows the maximum sulfur limits around the world in ppm. It can be clearly seen that generally, European fuel has low sulfur concentrations of around 10ppm. In comparison, large parts of Asia, Africa and South America have much larger sulfur contents in crude oil of up to 2500ppm.

When comparing Table 2-1and Figure 2-5,a correlation can be seen between sulfur limits in fuel oil and the sulfur contents of the crude oil exported from certain geographic locations. For example, Table 2:1 shows that Venezuela has very high sulfur content in its crude oil. This could explain why the environmental regulations there are not as strict as other countries. This could be due to the cost of sulfur removal from crude oil which is high.

As the world is pushing towards being more environmentally aware to the risk of global warming and climate change, ultra-low sulfur fuel is a very important part of contributing

29 towards this. Sulfur removal processes therefore must expand and progress in order to produce ultra-low sulfur fuel at a low cost. In this study, the crude oil used is from the Stanlow Oil Refinery in UK, and it has a sulfur content of 2200ppm, which must be reduced to 10ppm in order to meet the Euro V regulations (Neste Oil).

2.5.1. Approach for Limiting SO2 Emissions Limiting SO2 emissions, caused by fuel burning, requires the use of low-sulfur fuel, removal of sulfur in the feed, and appropriate technologies to control combustion and emissions. In 1990, according to Euro IV regulation, Sweden started to offer low sulfur diesel. In 2003, minimum aromatic diesel and zero sulfur were obtained for use in highly contaminated and confined regions (Lloyd et al., 2001). In the same year, it was planned that by the end of 2007, the sulfur content in non-road diesel fuel should reduce from 3400ppm to 500ppm by the United States Environmental Protection Agency (US EPA)(Song, 2003). Since 2005, based on the agreement with the Euro IV emission regulation, 50ppm of sulfur in diesel fuel for highway vehicles were allowed. There were attempts to obtain clean sulfur diesel (CSD) since 2006 in the United States, where most of the transportation fuel obtained <15ppm CSD limits. Non-highway vehicles such as off-road vehicles achieved the requirement for <500ppm clean transportation fuel limit before 2008. In 2009, there was a standard limit for both highway and non-highway vehicles according to Euro V’s aim for clean sulfur diesel (CSD); with a limit of 10ppm and they were compulsory to achieve the CSD requirement in 2012. (Barrett et al., 2012); (Lloyd et al., 2001).The European Union has established a policy for transportation fuel to reduce sulfur content by 2009 from 50ppm to 10ppm (Song, 2003) . Moreover, different countries in Eastern and Central Europe and other Asian countries and USA, Australia, New Zealand, Taiwan, Mexico, Singapore, and Hong Kong have established policies to reduce sulfur limits in transportation fuels. (Kilbane Ii, 2006, Zietsman, 2007). The International Council on Clean Transportation (ICCT) (2015), as shown in Table 2-2 a G20 briefing paper reviews today’s world-class emission standards (Kodjak, 2015).

30 Table 2-2 Summarizes the prevailing world-class emission standards (with permission ICCT) (Kodjak, 2015)

2.6. Dibenzothiophenes (DBT) DBT is the most widely used model sulfur compound in bio-desulfurisation studies (Mohebali, 2008). It has also historically been a good model compound for diesel BDS because alkylated DBTs are some of the main classes of sulfur compounds present in this oil stream (Neste Oil). Thiophenes such as DBT form the bulk of crude oil sulfur contents. Petroleum products are also produced from crude oil (Kilbane, 2004). DBT is generally found in an oil-contaminated setting, making it ideal for a study on degradation (Yu et al., 2006). Sulfur compounds in petroleum products exist as DBT up to 60% in content (Monticello, 1998). It is non-toxic to human health and is non-mutagenic and, therefore, hazards in the experimental part of the researches are minimized (Pokethitiyook, 2008). In addition, DBT is available as a pure, rich compound. This has enabled its wide use in biodesulfurization research. There is a lot of polyaromatic sulfur heterocycles in diesel with different alkyl groups that give various isomers. DBT is one of these isomers that can be used as a representative isomer (Monticello, 1998). The higher boiling aromatic compounds are the significant compounds of concern because of the difficulty to remove

31 from crude oil but Dibenzothiophene (DBT) is the aromatic organic sulfur found in crude oil with reasonable boiling point above 200oC ( Eisele,Walqui, and Kawatra, 2013).

Emissions from fuel combustion include sulfur dioxide, which is the main cause of acid rain and air pollution. During the refining process of crude oil, polycyclic aromatic sulfur- containing heterocyclic (PASH) compounds tend to increase slowly, in particular DBT content (CX-DBT). For example, 4,6-dimethyl dibenzothio-phene (4,6-DMDBT) is recognised as the main organosulfur compound, and the total sulfur content in diesel catalysis is restricted to 500 mg L-1. Due to increased environmental concerns, there has been an increased interest in the efficient removal of sulfur in CX-DBT in the petroleum refining process in recent decades.

As mentioned above, one of the methods for sulfur-removal from fossil fuels is biodesulfurisation. Organic naturally-occuring sulfur in crude oil can be manipulated by microbial activity in order to target sulfur removal. Biodesulfurization is generally perceived to be a good method as it has low costs, it is environmentally friendly, and requires moderate reaction conditions. Therefore, it is recognised as an alternative technology in sulfur-removal.

The traditional hydro-desulfurisation method costs much more than the biological desulfurisation , it needs high temperature and pressure in the presence of hydrogen. Alternatively, it is estimated that a biodesulfurization plant is two thirds of the hydro desulfurisation plant size. Also, operating costs are reduced by 15%, and carbon dioxide emissions decreased by 70-80% in biodesulfurization plants(Feng et al., 2016).

Biodesulfurization uses ring-destructive pathways to metabolise DBT. The 3-ring- destructive metabolic pathways have been recognised as Kadama pathway; C-S reduction pathway; and the specific 4S pathway (Abin-Fuentes et al., 2013). Section 2.8. Biodesulfurization Pathways explains each pathway in more detail. Since the conventional hydrodesulfurization of crude oil distillates in the diesel range, it usually leaves significant traces of alkylated-DBT compounds. It also works as an ideal compound in the biodesulfurization pathway of sulfur degradation (McFarland, 1999).

32 Several studies have reported the efficient degradation of DBT by some strains of bacteria (Soleimani et al., 2007). It has been shown that the sulfur atom is oxidizable to sulfoxides and sulfones (Omori et al., 1992). In other cases, these bacteria can grow on DBT utilizing its sulfur and carbon content for energy. It has been observed that the sulfur is converted to sulfite and then oxidized to sulfate, although this mechanism remains unclear (Izumi et al., 1994).

2.7. Methods of Desulfurization There are various desulfurization methods to remove sulfur from fossil fuels. These include desulfurization by ionic liquids (ILs), hydrodesulfurization (HDS), oxidative desulfurization, adsorption process, reactive adsorption, physical adsorption, and recently the use of applied biodesulfurization. HDS is considered to be the most important desulfurization method.

2.7.1. Desulfurization by Ionic Liquids (ILs) Ionic liquids are a salt in liquid phase. They have immeasurable vapour pressures at points lower than their decomposition temperature. The choice of organic ions to form ionic liquids is very important for desulfurization (Soleimani et al., 2007). For example, imidazolium ions with larger alkyl substitution groups, such as DBT, single β, and di-β methylated DBTs, have been reported as ionic liquids to remove (Huang et al., 2004). Also, the size of anions plays an important role in the extraction of DBT from the oil – phase. For example, large-sized anion such as [OcSO4] are able to extract DBTs more − - effectively than small-sized anions such as [PF6] or [CF3SO3] . It is considered that the size of the substituted alkyl group plays a significant role and that certain sizes may render the solvent ineffective (Soleimani et al., 2007); (Ibrahim et al., 2017).

2.7.2. Hydrodesulfurization Hydrodesulfurization is a commonly used technique to remove sulfur in refineries. It is the catalytic conversion of sulfur in sulfur-containing compounds over a CoMo/Al2O3 or

NiMo/Al2O3 catalyst at high temperature and pressure in the presence of hydrogen gas to hydrogen sulphide (H2S), whereby it is afterward reduced to elemental sulfur by an air oxidative process (Soleimani et al., 2007), (Kawaguchi et al., 2012). An example of the hydrodesulfurization direct mechanism of dibenzothiophene is shown in Figure 2-6. For this process, the temperature and pressure are usually within the range of 200 to 425oC and 150 to 250 psi H2 respectively. Recent research (Moonen et al., 2017) reports that to achieve the target sulfur contents, the required temperature should be at least 335oC.

33 Therefore, this process is slightly costly although it has been used for a long time. It is considered cost-effective for removal of sulfur from fossil fuels. However, it is too costly for desulfurization of refractory sulfur compounds (i.e. 4-alkyl- and 4, 6-dialkyl- dibenzothiophenes) (Labana et al., 2005b, Monticello and Finnerty, 1985).

Figure 2-6 Direct mechanism for the hydrodesulfurization of dibenzothiophene (Mudt et al, 2006). 2.7.3. Oxidative Desulfurization Unlike in the hydrodesulfurization process, whereby sulfur is removed from sulfur- containing compounds in the form of hydrogen sulphide, sulfoxides and sulfones are produced in oxidative desulfurization. These compounds are more polar than hydrocarbon compounds and thus can facilitate efficient removal of sulfones through solvent extraction of adsorbed solid materials. Oxidizing equivalents are supplied from chemicals such as hydrogen peroxide, organic hydroperoxides, and molecular oxygen (Yaqoub, 2012). These reactions require the use of catalysts to speed up the process of oxidation. Essentially, many forms of catalysts have been developed for this purpose. The dependence on oxygen or oxidizing agents allows the process to take place under mild conditions of low temperature and pressure, which may result in a cost-effective conversion process. However, problems of catalyst recovery, enormous quantity of oxidizing agents, poor selectivity, and low activity prolong reaction times (Al-Shahrani et al., 2007).

2.7.4. Physical Adsorption This method makes use of sorbent materials to remove compounds like mercaptans , sulphides, and thiophenes under mild conditions (Salem, 1994). The chemical identity of the sulfur compounds remain intact in the process, i.e. they are not changed but are only removed. The strength of adsorption is in the order 4,6-DMDBT > DBT > BT > 2-methyl thiophene > thiophene, for thiopene-containing materials. Even though this method is simple, the uptake of sulfur compounds depends on the nature of the sorbent material.

34 Also, aromatic compounds and alkenes compete for the sorbents if present; thereby reducing the efficiency of sulfur uptake. Thus, it is beneficial to use pre-adsorbents to remove these competing compounds so as to improve sulfur uptake (Yang et al., 2005, Hernández-Maldonado and Yang, 2003).

2.7.5. Biodesulfurization This process uses biological agents for sulfur removal. This bioprocess takes place under ambient temperature and pressure; thus requiring less energy. Hydrogen gas is not needed. Therefore, it is more economical and less technically demanding. There are also low emissions and no production of undesirable by-products (Monticello, 2000). If enzymes are the biocatalysts, then a high selectivity is obtained which is an advantage over some chemical methods of desulfurization. Despite these advantages, there are obvious concerns over commercialization of the biodesulfurization process in that biocatalyst activities are significantly lower than the rate of chemical conversion. Also it is not easy to maintain biological activities for long periods of time (Mohsen Sohrabi*, 2012, Labana et al., 2005b, Abin-Fuentes et al., 2013, Soleimani et al., 2007). The most important disadvantage of biodesulfurization using enzymes or microbial cells is that unless they are immobilised, they are lost during the process and hence there is a need for continued replacement of these biological agents. Immobilisation on the other hand, introduces mass transfer limitations because of their very small size, bacterial cells and/or enzymes need to be immobilised or encapsulated in gel-like matrices. Nevertheless, there has been significant interest in biodesulfurization research.

2.8. Biodesulfurization Pathways Biodesulfurization (BDS) is the process of using biological organisms to remove sulfur from a substance. Organisms such as fungi and bacteria can be used for this process. The key breakthrough for the use of bacteria as an organism for biodesulfurization was the discovery of a metabolic pathway of Rhodococcus erythropois strain sp. IGTS8. The bacterium was found in coal deposits. It was discovered that it removes sulfur from polycyclic aromatic sulfur compounds; without loss of coal fuel value. This natural breakthrough was also found in other organisms such as Rhodococcus erythropois strain sp. D-1, H-2, Corynebacterim sp. strain SY-1, and the Gordona sp. Strain CYKS1. All were identified to have the DBT-desulfurizing activity. IGTS8 is the most widely studied of these organisms (Ohshiro et al., 2005).

35 In about two decades, the quality of crude oil has been an important field in developing biotechnology. Many studies have been focused on removing the sulfur from fuel without reducing the calories by microbiological/biochemical methods, some examples for each method will be detailed later in this section. There are metabolic pathways for biodesulfurization to eliminate sulfur. There are mechanisms of DBT degradation by anaerobic biodesulfurization via Reductive C-S bond cleavage and by aerobic bacteria such as: Kodama, 4S, and Angular deoxygenation (Mohsen Sohrabi, 2012).

2.8.1. Kodoma Pathway The Kodoma pathway The Figure 2-7 below is the first reported aerobic pathway of DBT biodesulfurization (Kodama et al., 1970) by microorganisms Pseudomonas jijani and P. abikonesis, without removing sulfur (McFarland, 1999); (Gupta et al., 2005). In this pathway, the ring cleavage of one aromatic ring in sulfur compounds occurs by oxidative C-C cleavage, during initial oxidative reaction. The Kodama pathway has three main steps: hydroxylation; ring cleavage; and hydrolysis.

The Kodama pathway has some disadvantages • The carbon skeleton of sulfur compounds is broken by the bacteria which make the final fuel product , with low calories which means it is not commercially useful for the petroleum industry(Campos-Martin et al., 2010). • The Kodama pathway is not complete ring destructive of DBT without removing sulfur. • Anaerobic biodesulfurization via the Kodama pathway is caused by hydrocarbon ring cleavage and destruction in DBT. Moreover, after finishing biodesulfurization through this pathway, formal benzothiophen remains and sulfur specific releasing mechanism is not executed at the end of this pathway. • In fact, the Kodama pathway is not a preferable mechanism of biodesulfurization, because it produces water-soluble sulfur compounds, which are not burnable, at the end of this process. This causes a decrease in caloric value and the thermal unit of fuel is reduced which is not acceptable economical (Egrorova, 2003 ).

The Kodama pathway has been applied to many bacteria such as Pseudomonas jijani and P. abikonesis ( Kodama et al., 1970) and P. putida (Gupta et al., 2005 ) and also with fungi such as Cunnighamella elegans (Crawford and Gupta, 1990) cytochrome P-450 (Sariaslani and Dalton, 1989) and Pleutoris ostreatus (Bezalel et al. 1996). In these

36 examples, the intermediate component was DBT-5-oxide and the end product was DBT-5, 5-dioxide. However, with Rhizobium meliloti and Beijerincka 3-hydroxy-2-formyl benzothiophen (HFBT) was the end product (Frassinetti et al., 1998); (Laborde and Gibson, 1977).

The Figure 2-7 Kodama pathway of DBT degradation (Srivastava, 2012)

2.8.2. The 4S Pathway The 4S metabolic pathway is the most common biodesulfurization pathway, well characterized at the biochemical and molecular levels. The specific 4S pathway is used to

37 remove DBT by catalysing the C-S bond and breaking to form 2-hydroxybiphenyl and water-soluble sulfates without destroying the benzene ring structure. It is recognized as the most valuable of all the desulfurization pathways as it does not reduce the caloric value of the fuel which would in turn have no effect on the value of oil. It is important to understand the 4S metabolic pathway and its mechanisms used in the biodesulfurization of strains, in order to efficiently promote commercial biodesulfurization using this method to ensure it is economically viable. The 4S pathway was discussed firstly by Kilbane (Kilbane Ii, 1990). It can occur when the sulfur atom is released from the DBT molecule by oxidative desulfurization, while the hydrocarbon structure of the DBT molecule is protected (Bressler et al., 1997). Its 4S namesake is due to it involving four intermediate sulfur compounds and four enzymes, which each contribute to the pathway steps as catalysts. These catalysts are named DszC, DszD, DszA, and DszB. The pathway proceeds via two cytoplasmic monooxygenases (DszC, DszA) supported by a flavin reductase (DszD) and a desulfinase (DszB) (Sousa et al., 2016). They attack the sulfur part of DBT selectively and release the sulfur from the hydrocarbon as shown in Figure 2-8. These benefits have recently caused researchers to focus on the 4S pathway over the Kodama pathway, as it is able to keep the carbon structure intact after cleaving the carbon-sulfur bond in DBT during the oxidative desulfurization pathway (Abin-Fuentes, Mohamed et al. 2013). The 4S pathway, as shown in The Figure 2-7 can be completed in three stages from the chemical reaction point of view: activation of thiophene ring for cleavage; the aromatic sulfinate formation; and sulfinate group removal. The final products of the 4S pathway of DBT desulfurization are HBP and sulfate (Monticello, 1998); (Gray et al., 1996); (Labana et al., 2005b). DBT monooxygenase (DszC) catalyses the conversion of DBT to DBT sulfoxide (DBTO) and DBT- sulfone (DBTO2). DBTO2 monooxygenase (DszA) catalyses the oxidative C-S bond cleavage producing 2-(2′ -hydroxybiphenyl) benzene sulfinate (HBPS). DszB, an aromatic sulfinic acid hydrolase, affects a nucleophilic attack of a base-activated water molecule on the sulfinate sulfur to produce 2-hydroxybiphenyl (2-HBP) as a dead-end product and sulfite as bio-available sulfur for microbial growth. DszD delivers the reducing equivalents (FMNH2) needed for the functionality of DszC and DszA. The oxygen atom incorporated at each step of the pathway is derived from atmospheric oxygen (Ismail et al., 2016b).

38 Conversely, the disadvantage of the 4S pathway is that it is energetically expensive. This is because the carbon skeleton is not mineralized in order to get back the energy invested. The use of this pathway has been proposed for the desulfurization of petroleum in production fields and refineries (McFarland, 1999). Biodesulfurization has yet to be commercially applied, despite all the progress in the last 20 years. One reason is due to the stability and efficiency issues of the microbial biocatalyst (Monot & Warzywoda, 2008). Most of the research undergone on microbial desulfurization has used axenic cultures, which contain only one species, variety, or strain of a microorganism. However, it is worth researching the biodesulfurization capabilities of microbial consortia, to benefit from the cooperative microbe-microbe interactions (McGenity et al., 2012; Mikesková et al., 2012). Recently, engineered synthetic bacterial consortia have shown enhanced desulfurization abilities of oil sulfur compounds (Martínez et al., 2016). Despite the massive researches of using various strains of microorganism in the desulfurisation process. However, bacteria have not been industrialised for several reasons, such as the expense incurred from using bacteria, and the inability of the aqueous phase to form a mixture with the oil phase, without a surfactant present. This leads to enhance the importance of attempt plant cells in biodesulfurization, the novel process in this project. In the following Table 2-3 ,2-4 ,2-5 and 2-6 , some quantitative information on biodesulfurization by various bacteria is compiled but crucial information such as cell concentrations is lacking in order to calculate the specific biodesulfurization rates for quantitative comparisons.Researchers have considered biodesulfrization as an important choice for treatment of removing sulfur by 4S pathway from sulfur compounds in aqueous medium such as DBT as model of sulfur source as shown in Table 2-3 . On the other hands, different crude oil fractions can be treated by 4S pathway, which maintaining the caloric value of fuel not like other desulfurization pathway (Mohsen Sohrabi, 2012). This has been examined in various of bacterial strains, as has reported in the tables below. Table 2-4 illustrate the BDS capabilities of some microbes can be observed in with the model oil used and the percentage of sulfur removed from the oil recorded (Srivastava, 2012). In addition, some examples of biodesulfrization by some bacteria strains and the percentage of sulfur removed Crude oil fractions and their sulfur contents shown in Table 2-5 (Mohebali and Ball, 2008).

Table 2-3 Some examples of microorganisms which can desulfurize the DBT in the 4S pathway.

References optimum Name of Initial DBT Day of DBT uptake the use the temperatures, species DBT consumption incubation rate (ppm/h) same (ppm) (%) ◦C bacteria (Gallagher et al., 100 R. rhodochrous 1993)Kayser, 33166.8 95% 1 day 30 - 50 1312.852 Cleveland et IGTS8 2-HBP al. 2002) (Caro et al., 2007) Rhodococcus 28 - 35 (Mohamed et 92.13 100 56 h 1.645 sp. strain SA11 al., 2015) Kirimura, Bacillus Furuya et al. 2001 subtilis WU- 147.408 100 2h 50 73.704 50 C S2B (Labana et al., 2005a) Arthrobacter (Dahlberg et 55.278 100 30 h - 1.8426 K3b al., 1993) Mycobacterium 65% to Kayser, sp. GTIS 10 = 2-HBP Cleveland et 7370.4 48 h 25 - 75 99.807 al. 2002; M. phlei 3.3% to (Kilbane, GTIS10 Biophenol 2004) M. pheli WU- (Furuya et al., 99.5004 100 4 days 20 - 23 1.036 F1 2001) Enterobacter (Papizadeh et 147.408 64% 10 days 30 0.393 spp. NISOC-03 al., 2017) Sulfolobus (Gün et al., 55.278 88.5% 10 days 75-85 0.203 solfataricus P2 2015) Rhodococcus (Davoodi- erythropolis 552.78 100 10 h 30 55.278 Dehaghani et SHT87 al., 2010) Rhodococcus rhodochrous (MTCC 3552) Arthrobacter sulfureus (MTCC 3332) (Bhanjadeo, 92.13 99% 10 days - 0.380 Gordonia 2018 ) rubropertincta (MTCC 289) Rhodococcus erythropolis (MTCC 3951)

40 Table 2-4 Shows the biodesulfrization capabilities of various microorganisms optimized conditions

(Srivastava, 2012).

Sulfur Sulfur Process Sample Model Oil Microorganism System Conc. T (°C) Removal (ppm) %

4S DBT hexadecane Gordonia Batch 100[32 30 90% alkanivoransRIPI 0] 90A

4S DBT Tetradecane Mycobacteriumg Fed 200 40 99% oodii X7B Batch

4S DBT Hexadecane Rhodococcus Batch 100 30 80% erythropolisIGTS 8

4S DBTs n-heptane Gordonia Batch 100 35 63% alkanivoransstrai n 1B

4S DBTs n-tridecane Bacillus Batch 100 50 50% subtilisWU-S2B

BDS DBTs n-tridecane Mycobacteriump Batch 150 50 99% hlei WU-F1 BDS DBT n- Rhodococcussp. Batch 1000 30 75%

Hexadecane strain P32C1 4S DBT Hydrodesulf Mycobacteriumsp Batch 535 45 86% urized diesel . X7B

BDS DBT Ethanol Microbacteriumst Batch 36 30 94% rain ZD-M2 BDS DBT n- Pseudomonas Batch 500 31 74%

Hexadecane stutzeri UP-1 BDS DBT + Light gas oil Sphingomonas Fermen 280 27 94% 4,6DM subarctica T7b tor DBT 4S DBT n- Bacterium, strain Batch 100 30 77%

Hexadecane RIPI-22 BDS — Hydrodesulf Pseudomonas Fermen 591 30 47% urized diesel delafieldii R-8 ter oil

41 Table 2-5 Biodesulfrization activity by some bacteria strains and the percentage of sulfur removed from Crude oil fractions and their sulfur contents (Mohebali and Ball, 2008).

Table 2-6 Desulfurization of Petroleum by Different bacteria strain (Esmail Alkhalili et al., 2017).

Figure 2-8 The 4S pathway of DBT biodesulfurization (Bhanjadeo et al., 2018).

2.8.3. Angular Deoxygenation Through Oxidative C-C Bond Cleavage and DBT Mineralization

The sole source of C, S, and energy in this pathway is DBT is shown in Figure 2-9 .Bacterial degradation of DBT via angular deoxygenation starts on the angular position adjacent to the sulfur atom (Hideaki Nojiri, 2001). DBT-sulfone, DBT-sulfoxide and benzoic acid were revealed as metabolic intermediates of DBT degradation (van Afferden et al., 1990). The C-S bonds are cleaved during the degradation of DBT by this pathway; sulfite was released in stoichiometrical amounts and was additionally oxidized to sulfate. In this pathway, DBT was oxidized by monooxygenase to form DBT sulfone; angular dioxygenase attacked the last compound, leading to the formation of 2, 3-dihydroxy biphenyl-2-sulfinate. The ring fission of this product is degraded to sulfite and benzoate by Meta cleavage, which is further metabolized to TCA cycle metabolites. Therefore, a complete mineralization of DBT with the release of S, and then sulfite is oxidized afterwards to sulfate. Bactreia Arthrobacter sp. (Dahlberg et al., 1993) and

43 Brevibacterium sp.(van Afferden et al., 1988) are two of the most important bacterial species that attack DBT via angular deoxygenation. The same pathway has been reported with Pseudomonas C18 (Campos-Martin et al., 2010; Denome et al., 1993 ).

Figure 2-9 Overview of the bacterial degradative pathway for DBT via angular dioxygenation. The structures shown in brackets have not been characterized. The arrows, with solid and broken lines indicate the enzymatic and spontaneous conversion, respectively (Hideaki Nojiri, 2001).

44 2.8.4. Anaerobic Biodesulfurization Via Reductive C-S Bond Cleavage Several researchers have reported that BDS could be operated by anaerobic and aerobic bacteria; the anaerobic BDS is carried out under sulphate reducing conditions. Therefore, the opportunity of non-specific oxic attack of hydrocarbons in diesel will be rejected under sulfate-reducing conditions. Furthermore, the anaerobic BDS does not release sulfate. However, the use of the anaerobic BDS is limited by the cost of establishing the reducing equivalent (generation of H2) and to maintain anaerobic conditions (Gupta et al., 2005). In addition, it has been found that the bacterium Desulfovibrio desulfuricans M6 accomplished desulfurizing of some organic sulfur compounds present in diesel, such as DBT under sulfate-reducing conditions (Kim et al., 1990); (Kim et al., 1995). In this pathway, DBT is used as an electron acceptor, which is converted and accumulated as 2-HBP, whereas H2S was the final product after reducing the sulfur: as shown in Figure 2-10.

Figure 2-10 Principle of reductive DBT desulfurization pathway by Desulfovibrio desulfuricans M6 , adapted from(Kim et al., 1990).

45 2.9. BDS and its Lack of Commercial Application in Industry

New sulfur-removal processes are constantly being studied to identify new ways to efficiently remove sulfur from crude oil. This is due to increasing concerns of sweet crude oil reserves which are decreasing. The result is increased use of sour crude oil, which has significantly higher amounts of sulfur. In addition to this, there are stricter environmental regulations because of high volumes of sulfurous gases emitted into the atmosphere.

As sour crude oil contains higher sulfur content, and is being used more widely, there is a demand for innovative separation methods for sulfur-removal. As mentioned earlier, HDS is a more expensive method than BDS. It is also not able to fully remove DBT in crude oil, which is the most common sulfurous component. As a result of this, oil refineries have attempted to incorporate BDS into desulfurization processes in order to improve separation techniques. However, this has not been implemented commercially yet. The reason for this is due to the difficulty in acquiring accepted industrial results. This has caused an increase in research; dedicated specifically to achieving large-scale experimental results (Boniek et al., 2015).

One of the biggest challenges faced in the attempt to implement BDS industrially has been the cost of growing micro-organisms in culture media. This could be overcome by using carbon sources obtained from agricultural products. Another significant expense is the cost of the micro-organisms themselves. It has been suggested that this cost can be reduced by immobilizing the cells onto luffas or the plant root itself (Frassinetti et al., 1998). In addition, another challenge faced in the industrial application of BDS is the high water to oil ratio, which should be minimized to decrease water separation and disposal costs (Foght, 2004).

To overcome the challenges of using BDS alone, Li et al. (2009) investigated a possible alternative of combining two desulfurization methods using adsorption and biological agents. Firstly, sulfurous compounds are adsorbed onto the adsorbent, which is then bio- regenerated using microbes. This study was conducted using DBT as the model sulfur compound and the biological agent used was bacterial P- delafieldii R-8 strain. The study found that the bacterial strain used had a similar particle size to the adsorbent, therefore, iron oxide nano-particles were required to modify the bacterial cells to facilitate the separation of the cells from the regenerated adsorbents. However, this process requires

46 further research to show crude oil desulfurization capability of the system to obtain ultra- low sulfur content (Li et al., 2009).

A study by Agarwal and Sharma (2010) showed that BDS can occur efficiently under anaerobic conditions by following the BDS process with oxy-desulfurization and then reactive adsorption. This method used both heavy and light crude oil. The results obtained indicated a 95.21 wt. % sulfur removal from the heavy crude oil and a 94.3 wt. % sulfur removal from the light crude oil. These results are very promising as the great majority of the sulfur was removed from both crude oils, and more so in the heavy crude oil which has a larger thiophenes content, which are characteristically more difficult to remove.

A more attractive option is to employ a small-scale BDS mechanism which can be used to HDS-treated crude oil, rather than a complete industrial-scale BDS process which may not lead to any economic gain. This alternative could also satisfy the need to adhere to environmental regulations. However, further research must be completed in order to explain the mechanisms and pathways of sulfur-removal by micro-organisms, the transport rate of sulfur from the oil to the biological agent membranes and the recovery of the bio-catalyst. Further study could also be carried out on the use of plant cells as a biological catalyst, instead of microbes, as this could be instrumental to the application of commercial BDS.

47 Chapter 3 Plant Cell Culture in Biotechnology

Most of the researchers focused on using microorganisms for biodesulfurization as summarised in Chapter 2. However, it has some disadvantages which can be avoided by using plant cell cultures in the biodesulfrization process. For example, microorganisms are not as easy as plant cells to harvested and scale up due to their small size. Moreover, microorganisms can be affected easily by the environmental factors (temperature, acidity, energy sources, and the presence of oxygen, nitrogen, minerals and water all affect bacterial growth). Due to the microorganisms’ short lifecycle, they cost more than plant cells to reuse; for instance, in the bioreactor. However, the novelty in this research work reported here is that plant cells are used instead of microorganisms, specifically cells of Arabidopsis thaliana, Armoracia rusticana and Nicotiana tabacum.

3.1. Plant Cell Culturing Protocols in Vitro In vitro plant cell cultures under controlled conditions are more beneficial than the cultivation of the whole plant for research. The establishment and development of plant cell cultures started a long time ago. For instance, in 1902, plant cells were first cultured Haberlandt (1902). The cells did not divide but he succeeded to isolate and keep them alive. The first suspension culture was made by Muir et al. (1954). Preparation of the medium for plant cell growth is the same as that for microbial media. Four different types of compounds should be provided; carbon source, inorganic salts, organic supplements and trace elements (Mavituna et al., 1988),(Vivanco, 1992). Growth regulators in photo-hormones are important for plant growth medium in vitro at a small amounts of around 10 mM for maintenance. There are 5 classes which are important in helping the plants retain their green colour for a long period of time. Growth regulators promote cell division and prevent flowers from appearing in the cell cultures. Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) and cytokinins such as benzylaminopurine (BAP) and kinetin. In this project, a specific amount of each hormone was used, as mentioned in Chapter 4 (Dixon and Gonzales, 1994); (Gaspar et al., 1996).

Reasons that Make the Use of the Plant Cell Culture System Beneficial Over the Use of Whole Plants • Economically, biomass can be re-used. The cells are not lost but remain in the bioreactor and are retrievable from the medium. Therefore, it is cost-saving. • It allows the use of expensive but efficient fermenters of high biomass / low volume vessels (Dixon and Gonzales, 1994). • Controlled growth processes and conditions can reduce the cost of labour. For example, a continuous process in automated equipment can save cost and yield more products. • The cells can be separated physically from the medium (Mavituna et al., 1988). • The climate and soil condition are no longer a significant problem; therefore, any compounds or products can be produced without depending on environmental conditions. Moreover, chemical and environmental factors can be easily controlled and made constant with little effort. • Better control can prevent contamination thereby ensure working in sterile atmosphere devoid of microbes and insects (Mavituna et al., 1988). • It allows the researchers to study the behaviour of any parts of a plant directly. • With artificial culture media, any reaction of target cell material can be investigated with any selected factors or components (Neumann et al., 2009).

However, uses of plant cells cultures are not without high risk of contamination, and there are difficulties in extrapolating results based on tissues cell cultures to explain the same phenomena that happen in the intact plant. Moreover, it is almost impossible to replicate in situ conditions in plant cell cultures (Neumann et al., 2009).

Many factors should be taken into account when working with plant cell cultures. The most important are environmental and biological controls. This means there are laboratory requirements that must be followed before experiments are started which may hinder growth and productivity of the cell culture.

A. Environmental controls (light, pressure, humidity, nutrients, constant temperature in the room or for growth of callus cultures, in the incubator, orbital flask shaker, a sterile area, room or air cabinet, medium preparation should be done in a separate area of the whole lab,

49 differentiation of cultured cells, cells growth and secondary metabolism can be effected by plant growth regulators). b. Biological controls of tissue growth, morphological differentiation, and control of changes or variations in biological activities among others (Barz et al., 2012). It is important that the cells and the products in the growth medium are safe from external contamination by microbes or any environmental changes such as temperature, pH, pressure, and humidity. Therefore, the experiments should be carried out under sterile but special living conditions in order to be able to produce a growth curve (Karl-Hermann Neumann and Imani, 2009).

3.2. Plant Cell Culture Applications When grown in-vitro, plant cell cultures are unaffected by geographical and seasonal variations, as well as other environmental factors. Compared to whole plants, other advantages include their enhanced uptake of media nutrients and relative ease to harvest. Genetically identical copies of the plant can cultivated in the form of plant cell and tissue cultures to produce compounds such as flavours, fragnances, pharmaceuticals and pigments in the absence of pollinators (Nichodemus, 2017); (Kieran et al., 1997).

On the other hand, there are some disadvantages of using plant cell cultures. Firstly, aseptic conditions are required, as well as exceptional care when handling as cultures so that they do not get contaminated. There is also a possibility that metabolites produced in the cells could block the vacuole. This could be overcome by using an electric pulse, pressure or UV radiation to manipulate the cell membrane permeability so they can be secreted into the medium (Nichodemus, 2017).

Another drawback of using plant cell cultures is the lack of information on the biosynthetic pathways of plant metabolic products; therefore, it is difficult to improve their production. Finally, there is a risk that cell characteristics may change after a period of continuous growth, as an initial cell population may exhibit different characteristics to new cell growth (Nichodemus, 2017). Another reason which makes the analysis challenging is aggregation. This is a common phenomenon which happens when cells division process after failing to separate. Aggregation patterns, can be studied by using image analysis and sieving (Kieran et al., 1997);(Mavituna and Park, 1987). This phenomenon can be useful for the development of self-immobilization methods (Prenosil et al., 1987);(Hegglin et al., 1990).

50 The role of cell – cell interactions in undifferentiated systems has yet to be conclusively established. However, Schuler (1993) has indicated that the degree of cellular association can be affected by metabolite productivity and arising scale-up differences in aggregation patterns. Moreover, N. tabacum, which we are using it in this study, has been described as a highly-aggregated species of plant(Hashimoto and Azechi, 1988); (Hooker et al., 1990).

3.3. Plant Species Used in this Work The majority of crude oil from the Earth’s surface is formed from the remains of pre-historic algae and zooplankton (Dept, 2009).The dead organisms mix with mud and other elements under high pressure from the sediment layers above them. They are then heated to very high temperatures to form petroleum. Therefore, as crude oil originates from organic plant matter, this would indicate that plants would be efficient bio-catalysts for BDS as they have the enzymes needed for sulfur consumption, which is essential for plant growth. The plants which were tried are Arabidopsis thaliana, Armoracia rusticana (horseradish), and Nicotiana tabacum (tobacco).Horseradish cells have been found to show a peroxidase characteristic which indicates the presence of enzymes which catalyse hydrogen peroxide oxidation of substrates. This has been linked to plant defence against pathogens (Karthikeyan et al., 2006). Horseradish enzymes contain an accessible active site. Therefore they can use a large variety of organic compounds as electron acceptors and donors. This has resulted in peroxidase being used in disposal treatment systems such as the treatment of waste waters and conversion of toxic materials into less harmful compounds. Moreover, as seen in Table 3-1 below Arabidopsis is from the same family as Horseradish that means it should have the same enzymes which can catalyse hydrogen peroxide oxidation of substrates. As shown in Table 3- 1 ,Nicotiana tabacum cells shown in the table are from Solanaceae family, and not from the Brassicaceae family as Arabidopsis thaliana, Armoracia rusticana (horseradish). Nicotiana tabacum cells however, have similar peroxidase characteristics to those of horseradish and Arabidopsis thaliana cells, giving it possible bio-desulfurization abilities (Burbridge et al., 2006). The oxidative desulfurization process uses hydrogen peroxide as an oxidizing agent. Hydrogen peroxide reacts with the sulfur to produce high polarity sulfone which is easily removed. Therefore, the hydrogen peroxide produced in the Nicotiana tabacum cells could potentially react with the DBT in petroleum and act as a catalyst for the bio-desulfurization of crude oil.

Table 3-1:The plants taxonomy of the plant which were using in this study.

Kingdom: Plantae Kingdom: Plantae Kingdom: Plantae

(unranked): Angiosperms (unranked): Angiosperms (unranked): Angiosperms

(unranked): Eudicots (unranked): Eudicots (unranked): Eudicots

(unranked): Asterids (unranked): Rosids (unranked): Rosids

Order: Solanales Order: Brassicales Order: Brassicales

Family: Solanaceae Family: Brassicaceae Family: Brassicaceae

Genus: Nicotiana Genus: Armoracia Genus: Arabidopsis

A. rusticana A. thaliana N. tabacum

Species: Species Species:

3.3.1. Arabidopsis thaliana The main plant species used for the initiation and establishment of the cultures were Arabidopsis thaliana, which is called thale cress, or mouse-ear cress or simply Arabidopsis. Arabidopsis thaliana is an important plant which has been used extensively for studies involving genetics and cellular and molecular biology of flower producing plants (Lamont, 2014) due to its basic plant features and its rapid life cycle (Negrutiu et al., 1975). Due to its small size, it can easily be grown in large quantities in vitro studies and thus produces many seeds. In addition, its genome has already been sequenced and there are genetic maps that makes it easy to locate gene groups, it also has a low chromosome number, n=5. There are already available mutant seeds that grow at a rapid rate and are easily transformed by Agrobacterium spp, for gene insertion work (Cotter, 2005, Negrutiu et al., 1975). Despite these beneficial behaviors, the disadvantages are not properly documented.

52 Remarkably, the use of tissue culture procedures for Arabidopsis thaliana improves the transfer of genetic materials in processes such as fusion, transformation, and gene expression (Masson and Paszkowski, 1992). Arabidopsis thaliana cultures were developed to screen for a specific product; for example up taking and degradation of DBT by the plant cell culture. DBT is used as a model for the sulfur compounds present in crude oil, which causes pollution to the environment. Therefore, the breakdown or destruction of DBT to produce a safe and non-toxic compound will be studied (Yaqoub, 2012).

3.3.2. Armoracia rusticana (horseradish) Armoracia rusticana has also been studied in this research. It is a plant, which originates from South Eastern Europe and Western Asia, and is popular globally. It is particularly used in Japan for cooking as well as for herbal purposes. Horseradish Peroxidase is a versatile commercial enzyme which originates from horseradish plant. It is used extensively in molecular biology for antibody detection, and has become increasingly important in biochemical research. Horseradish grows up to 1.5 meters tall, and is cultivated for its large white tapered root. Horseradish roots are hard and the plant secretes enzymes when cut or grated. The secreted enzymes break down the sinigrin to produce allyl isothiocyanate, also called mustard oil. Once the plant is cut or grated, it should be used immediately or mixed with vinegar to prevent the roots from darkening. Plant growth may be affected by viruses, which can affect the yield. Armoracia rusticana cannot be propagated from seed due to its sterility, and instead is propagated by cutting the roots, and by tissue culture technology. The entire plant can be regenerated. Reports of the largest embryogenic callus were formed in the MS (Murashige & Skoog) medium formulation supplemented with glucose and plant growth hormones 2, 4-D, BAP and gellan gum as a solidifying agent. The secreted enzyme horseradish peroxidase has been found in the plant, and produced in the liquid cell suspension. It can be used as a substance to absorb DBT, and Gas Chromatography (GC-FID) can be used to analyse this by measuring the amount of components in the sample and their corresponding retention times (Shaaban, 2016, Krishnan, 2009).

3.3.2. Nicotiana tabacum (tobacco) Nicotiana tabacum history began in 1492, when Christopher Columbus discovered American Indians using the herb’s leaves for medicating illnesses. In 1536, European travellers to the New World carried the herbal knowledge that they had witnessed from medics and physicians in the Western Hemisphere with them. This knowledge spread across Western

53 Europe, resulting in Western European physicians adopting Nicotiana tabacum as medicine (Kishore, 2014).

Nicotiana tabacum is naturally cultivated in the American continent mainly South America, as well as Australia and the South Pacific. This suggests that South America (borders of Argentina and Bolivia) is where Nicotiana tabacum originates (Kishore, 2014, Dixon and Gonzales, 1994).

Currently, Nicotiana tabacum is a novel on its application in BDS and its potential in breaking down DBT, due to its favourable growth characteristics. Nicotiana tabacum has been shown to have a high leaf biomass with high growth rate index values in both light and dark conditions. These characteristics have made it more favourable than cotton, tomato, carrot, and soybean (Dixon and Gonzales, 1994).

A study by (da Silva Madeira et al., 2008) on the effect of horseradish on DBT showed that about 60% of initial DBT concentration degraded to produce 46% dibenzothiophene sulfone and 14% dibenzothiophene sulfoxide. The presence of these two final products shows that the BDS mechanism did not follow the preferred 4S metabolic pathway which could lead to a reduction in the economic value of the oil obtained after desulfurization. Nonetheless, these positive results with horseradish, in only a short span of time of 1 hour, provide motivation for using Nicotiana tabacum cells as it has a similar cellular characteristic to horseradish.

3.4. Common Enzymatic Approach in the different Species of Organisms (human, animal, plant and microorganism) which can Oxidise Sulphur Components Many different prokaryotes and eukaryotes are capable of oxidizing sulfur compounds such as DBT which exist in crude oil. If the same or similar enzymes involved in bacterial BDS pathways are found within cells of other organisms such as plants, then it is possible that they too can perform part or whole of the BDS pathway reactions.

In order to confirm whether a relevant enzyme is present in plant cells, the webserver (SwissTargetPrediction) can be used. For example, NAD(P)H dehydrogenase, quinone 1 (by homology) according to this webserver exists in Homo sapiens and other mammals such as cattle, Mus musculus and Rattus norvegicus (Gfeller et al., 2013). NAD(P)H dehydrogenase (oxidoreductase) exists within Arabidopsis thaliana, Horseradish (Yaqoub, 2012) and Nicotiana tabacum (Sparla et al., 1996). The sulfur oxidation systems in bacteria are widely

54 researched and documented; for example (Lee et al., 2006) which can be observed in section2.8. Biodesulfurization Pathways

The last enzyme in that pathway is DszB (2'-hydroxybiphenyl-2-sulfinate desulfinase); an aromatic sulfinic acid hydrolase. It affects the nucleophilic attack of a base-activated water molecule on the sulfonates sulfur to produce 2-hydroxybiphenyl (2-HBP) as a dead-end product and sulfite as a bio-available sulfur for microbial growth (Lee et al., 2006, Martínez et al., 2016). On the other hand, in plants peroxidases are instead used in the degradation of sulfur compounds. This can be confirmed by the presence of the Horseradish Peroxidase (HRP) enzyme in Horseradish plants (da Silva Madeira et al., 2008), which contributes to its biodesulfrization ability. The molecular docking simulation can be used to confirm that the whole pathway can take place in plant cells. More details are provided in section 4.1. Molecular Docking Procedure

3.5. The Effect of Oil Contacting Plants The effect of oil spillages on animal life is well documented. Since the aim of this research involved contacting plant cells with crude oil, it was necessary to investigate both in the literature and as part of the experiments; the effect crude oil might have on plant cells. In 2010 in the Gulf of Mexico, a BP oil spill occurred which was caused by an explosion in the Deepwater Horizon oil rig. Oil spills like this have a detrimental effect on plant life as they can destroy the plants ability to carry out major processes such as photosynthesis which will eventually stunt its growth. This is because the spilled oil coats the plant leaves for a long time; preventing them from receiving crucial sunlight required for photosynthesis and growth (Etkin et al., 2005).

In the US, approximately 200 million gallons of oil are disposed of improperly every year (Setti et al., 1996).

Since 1970, Baker suggested that oils could additionally reduce transpiration rates in plants (Baker, 1970). Transpiration is the evaporation of water from the plant leaves where it exits through the pores on the underside of the leaves and is released into the atmosphere in the form of vapour. Oil can potentially block the stomata and intercellular spaces in the pore reducing the rate of transpiration. This reduction in transpiration would in turn contribute to similar declines in the rate of photosynthesis causing a disruption in the chloroplast membranes (Boniek et al., 2015). Photosynthesis takes place within the plant cell, and uses

55 solar energy to convert water and carbon dioxide into glucose and oxygen. It can therefore be concluded that a reduction in photosynthesis would reduce glucose produced; which is an essential requirement for growth. In Table 3:2, the various plants that were used in studies of oil effects on transpiration are recorded along with the effect on both transpiration and respiration.

Oil can affect respiration in multiple ways, depending on the type of oil and plant. In general, the addition of oil causes an increase in respiration rate; due to mitochondrial damage which creates an uncoupling effect (Boniek et al., 2015). During respiration, glucose and oxygen react and release energy, carbon dioxide, and water in the process. Therefore, a higher respiration rate produces greater amounts of carbon dioxide, which would contribute to plant growth resulting in more increased photosynthesis. However, this contradicts the point made earlier about the prediction that a reduction in transpiration will consequently reduce the photosynthesis rate. It can be observed in, Table 3-2 , that the respiration increased for two of the plant-oil combinations and decreased for the last combination. Based on these results, there is no strong correlation found between the rate of respiration and transpiration when a plant comes into contact with oil. The results suggest that the relationship between the two rates is dependent on the plant type and the particular oil it comes into contact with. Consequently, an assumption cannot be made on how crude oil would affect Nicotiana tabacum plant cells without practically testing the combination of cells and oil.

Table 3-2 How oil coming into contact with whole plants affects the rate of respiration and transpiration (Setti et al., 1997, El-Gendy and Nassar, 2018)

Plant Oil Transpiration effect Respiration effect

Citrus Highly refined white oil Reduced Increased

Parsnip Petroleum naphtha Reduced Increased

Mustard Petroleum naphtha Reduced Reduced

3.6. Hairy Roots: Mechanism of Transformation Some plants in nature have fibrous roots. If pieces of these are placed in plant cell/tissue culture media they will produce callus which can then become plant cell suspensions. However, there is a procedure of initiating plant hairy roots which can be cultured and sub-

56 cultured as hairy roots by following what happens in nature when wounded plants or plant roots are affected by a soil dwelling bacterium, Agrobacterium rhizogenes. This process is depicted in Figure 3-1. Hairy root cultures are stable and grow fast with simple defined media. They have been the focus of interest by many researchers for the production of secondary metabolites.

Figure 3-1 Agrobacterium rhizogenes infection mechanism (Golikov).

They can produce higher levels of secondary metabolites than do cell cultures. The amounts produced are comparable to those of intact plants. The secondary metabolites may be useful as cosmetics, food and pharmaceuticals additives (Choi et al., 2008). They are a source of root-derived pharmaceuticals or as a model system to study biochemical processes (Smetanska, 2008). In addition, induction of hairy roots can help to take up certain chemical compounds in order to study differences in biosynthetic pathways that occur in different systems in vitro culture and plants (Kastell et al., 2013). Hairy root cultures are still a recent phenomenon. Therefore, the advantages of hairy roots are noted in many studies. In several aspects for new technology, they are trying to scale- up culture and product recovery (Choi et al., 2008); (Flores et al., 1987); (Giri and Narasu, 2000). Agrobacterium rhizogenes is a gram-negative soil bacterium that enhances the hairy roots growth in the plant cells. It produces hairy root disease. The hairy root phenotype grows

57 much faster than normal roots without needing an external supply of auxins. In addition, hairy roots are biochemically and genetically stable (Ramachandra Rao and Ravishankar, 2002), and synthesize natural compounds at levels comparable to intact plants (Flores et al., 1987);(Nam Il Park1, 2010).

Agrobacterium rhizogenes contains root inducing (Ri) plasmid which can be transformed into the genome of the infected plant Figure 3-1. The Ri plasmid first ensures it is integrated into the host plant genome and becomes stable after which the diseased phenotype begins to be evident in the plant. Invariably, the integrated Ri T-DNA takes over the regulation of endogenous hormones of the transformed tissue, which consequently leads to an increased growth rate of hairy root (Flores et al., 1987); (Nam Il Park1, 2010).

Finally, hairy roots can be grown as a packed column which is self-immobilised and in this was can be studied for the biodesulfurization of crude oil as discussed in Chapter 6 .

58 Chapter 4 Experimental Materials and Methodology

This chapter, starting with the molecular docking procedure, and then medium preparation and the methodology for running the experiments, will be explained which can then justify that Nicotiana tabacum has the same enzymes as 4S pathway (Figure 2-8) which occurs in the microorganizms used for biodesulfurization. The experimental procedures and techniques used will be explained in detail. Nicotiana tabacum (tobacco) was the chosen plant for the biodesulfurization process. However, two other plants were also tested, Arabidopsis thaliana and Armoracia rusticana (horseradish). The cultures were firstly initiated from seeds by germination but only Armoracia rusticana (horseradish) was initiated from explant, then callus initiation happened, followed by transfer of the callus to a suspension culture and then several subcultures of the cell suspension cultures continued bulking up until enough cells were available to start examining the plant cell culture growth with sugar and DBT analysis. As mentioned in Section 3.2. Plant Cell Culture , Nicotiana tabacum has a high tendency of aggregation that necessitates the use of the sieving technique to obtain fine cell aggregates. This in turn helps obtain the fresh and dry weight measurements more easily and more accurately. Crude oil was added after examining the plant’s ability to grow in the presence of DBT. The establishment of hairy root process will be explained later in this chapter. Sterilization is required in all the steps involved in this research in order to avoid contamination by microorganisms. This is because plant cell growth is slower than microorganism growth; thereby causing contamination problems. This can be avoided by doing the experimental work aseptically which can be achieved in a sterile laminar air flow cabinet. However, following the safety restrictions, the crude oil experiments should be done under the fume cupboard; it pushes the air upwards rather than the outwards toward the researcher as in the laminar air flow cabinet. The stock solutions should be ready before preparing the medium and then the equipment and the media should be autoclaved at 121°C and 15psi for 20-30 minutes. Moreover, the room where the cultures are incubated should be maintained at 25±1°C in the presence of light from fluorescent tubes at 2000lux (42μ mol m-2 s-1). The suspension cultures are placed on an orbital shaker at 110rpm. The safety aspects of this work are covered in the documents uploaded to CEAS OPRA website (https://intranet.ceas.manchester.ac.uk/OPRA/OPRAHome.asp)with RA7946, 8897, and CEAS FM 029 for Agrobacterium rhizogenes for the hairy roots protocols.

4.1. Molecular Docking Procedure

A literature search was carried out to find out Nicotiana tabacum, Arabidopsis thaliana and Armoracia rusticana (horseradish) plant enzymes involved in the pathway phenylpropanoid biosynthesis and in aromatic compound metabolism; including sulphur compounds oxidation. Plants contain NADP(H) dehydrogenases (NAD (P)H:(quinone-acceptor) oxidoreductase. NAD(P) H-QR, (EC 1.6.99.21) of Nicotiana tabacum.leaves and roots have been purified. Another enzyme ( in the used plant) that was found to be involved in the above pathway was Feruloyl-CoA thioesterase which is similar to Alfalfa feruoyl coenzyme A 3-O- methyltransferase, an enzyme that can oxidised sulphar containing compounds. Alfalfa feruoyl coenzyme A 3-O-methyltransferase is similar to an enzyme AtDHNAT1, a 1,4- dihydroxy-2-naphthoyl-CoA thioesterase from Arabidopsis thaliana. The Swissdock server provided by the Swiss Institute of Bioinformatics (www.swissdock.ch/) was used for the docking of the target ligands. SwissDock, a web service to predict the molecular interactions that may occur between a target protein and a small molecule.It Propose a binding mode for a ligand a generate a complex to perform subsequent calculations,create figures for your articles and design inhibitors for the target of your choice. SwissDock is software is based on EADock DSS alogarithim. (www.swissdock.ch/),(Grosdidier et al., 2011a),(Grosdidier et al., 2011b). The Swissdock server in this project was used for the docking of DBT, DBTO, DBTO2, and HBPS ligands to NADP(H) Reductase, Alfalfa feruoyl coenzyme A 3-O-methyltransferase, and AtDHNAT1, a 1,4-dihydroxy-2-naphthoyl-CoA thioesterase. The X-ray structures of NADP(H) Reductase (PDB ID:4hfm), Alfalfa feruoyl coenzyme A 3-O-methyltransferase (PDB ID:1sus), and AtDHNAT1, a 1,4-dihydroxy-2-naphthoyl-CoA thioesterase (PDB ID:4k02) were used for this work (Figure 4-1) . The figures were constructed using UCSF Chimera and another molecular display tool called Pymol.

Figure 4-1Cartoon representation of the X-ray structure of (a) NADP (H) Reductase (PDB ID: 4hfm), (b) Alfalfa feruoyl coenzyme A 3-O-methyltransferase (PDB ID: 1sus) and (c) AtDHNAT1, a 1, 4-dihydroxy-2- naphthoyl-CoA thioesterase (PDB ID: 4k02). In all three figures Helix, Beta sheet, and Loop. (PDB = Protein Data Bank).

To check the existence of identical or equivalent (isoenzymes) of bacterial biodesulfurization pathways in plant cells, the molecular docking steps followed are explained in detail below: 1- Searched the full name of the enzymes in Kegg (Kegg.com, last access 2018)that are equivalent to 4S pathway enzymes in bacteria (DszC= Dibenzothiophene monooxygenase, DszA = Dibenzothiophene sulfone monooxygenase, DszB =2'- hydroxybiphenyl-2-sulfinate desulfinase). From various databases (Brenda- enzymes.org),(https://enzyme.expasy.org/),(http://www.uniprot.org/)and (http://enzyme-database.org./) find out the equivalent enzyme in plant from these servers. DszC enzyme from bacteria the equivalent in all plants 4hfm (X-ray Crystal Structure of a NADP (H)), which can convert DBT to DBTO, DBTO to DBTO2 and DBTO2 to HBPS. But in different affinity and it is work better in the beginning of the pathway as(DszC) and DszA).The (DszB) is the enzyme which is work effectively in the last step in the pathway where the sulfur is released from DBT by convert 2HBPS to 2-HBP , the equivalent enzyme in each plant is 1sus ( Alfalfa feruoyl coenzyme A 3-O-methyltransferase) in Nicotiana tabacum ,4k02 (AtDHNAT1, a 1,4-dihydroxy-2- naphthoyl-CoAthioesterase) in A.thaliana and HRP (Horseradish peroxidase) in Horseradish. Depending of the finding enzymes the predicted biodesulfurization pathway in plant cells has drown as seen in the results and the efficiency of this enzymes will discussed as well. 2- Retrieved the enzyme structures from Protein data bank server (https://www.rcsb.org/).

61 3- Submit the legend (Sulfur compounds) and the enzyme to Swissdock server (docking simulation ) to obtain the docking simiulation output (www.swissdock.ch/). 4- The docking simiulation output was analysed by using the Swissdock server plug in UCSF Chimera and Binding energies [E (Kcal/mol)]and ∆G (Kcal/mol) and then used

the Equation ∆G = -RTlnKD to calcualte Dissociation constants (KDs) and then

convert KD to affinity constants (Ka= 1/KD).

4.2. Medium Preparation The chosen medium in this study is Murashige and Skoog (MS); as shown in Table 4-1, which was supplied by Sigma. Murashige and Skoog (MS) Sulfur free medium (SFM) was purchased from US Cassion labs (Table 4-1). Some of the medium components for plant cultivation such as kinetin, naphthalene acetic acid (NAA), and 2, 4- dichlorophenoxyacetic acid (2, 4-D), which are plant growth regulators are needed for the cells to grow in vitro. Both MS and SFM, when supplemented with a sulfur source such as DBT, which work well with the plant cells. MS medium is the commonly used plant culture medium (Dixon and Gonzales, 1994). Although according to the literature, different plant species may require different types of media, all the plants used in this research, Arabidopsis thaliana, Armoracia rusticana (horseradish) and Nicotiana tabacum (Nicotiana tabacum) can grow well in MS medium. Therefore, MS was used in this study. For preparing solid medium, a specific amount of agar should be added (Catherine et al., 1993), as shown in Table 4-2.

62 Table 4-1: The difference between normal and sulfur free Murashige and Skoog basal medium composition.

MS Concentration Concentration SFM Component Component (mg/l) (mg/l) Ammonium Nitrate Ammonium Nitrate 1650.00 1650.00 (NH4NO3) (NH4NO3)

Boric acid (H3BO3) 6.2 Boric Acid (H3BO3) 6.2 Calcium Chloride Calcium Chloride, 440 332.20 (CaCl2 · 2H2O) Anhydrous (CaCl2) Cobalt Chloride, Cobalt chloride 0.025 Hexahydrate 0.025 (CoCl2 · 6H2O) (CoCl2 . 6H2O)

Cupric sulfate (CuSO4 · Copper II Chloride, 0.025 0.013 5H2O) Anhydrous (CuCl2) EDTA, Disodium,

Na2EDTA · 2H2O 37.2 Dihydrate (C10H14N2Na2O8 . 37.260

2H2O) Ferrous sulfate Iron III Chloride, 27.8 16.220 (FeSO4 · 7H2O) Anhydrous (FeC_l3) Magnesium sulfate Magnesium Chloride, 370 142.930 (MgSO4 · 7H2O) Anhydrous (MgCl2) Manganese Chloride, Manganese sulfate 22.3 Tetrahydrate 19.79 (MnSO4 · 4H2O) (MnCl2 . 4H2O) Sodium molybdate Sodium molybdate 0.25 0.2500 (Na2MoO4 · 2H2O) (Na2MoO4 . 2H2O) Potassium Iodide (KI) 0.83 Potassium Iodide (KI) 0.8300

Potassium nitrate (KNO3) 1,900 Potassium Nitrate (KNO3) 1900.000 Potassium Phosphate, Potassium phosphate 170 Monobasic, Anhydrous 170.000 (KH2PO4) (KH2P O4) Zinc sulfate Zinc Chloride, Anhydrous 8.6 4.080 (ZnSO4·7H2O) (ZnCl2)

63 The preparation of the media should be accurate in terms of measurement and sterilization, while adding all the supplements with the agar in the required quantities as is shown in Table 4-2. The vessels, which contain the media, are placed on the magnetic stirrer and the rotation speed set to 80 rpm. This is to ensure that all the compounds are dissolved uniformly in the medium. After dissolving, the pH of the media should be adjusted to 5.8 (Dixon and Gonzales, 1994). Sodium hydroxide is the base, which can be added to raise the pH level, and hydrochloric acid is the acid used to decrease the pH level, in order to adjust the pH of the solutions. After that, the prepared medium is sterilized by autoclaving at 121°C for 20-30 minutes. The medium should be left in the laminar air flow cabinet to cool down to a hand-bearable temperature of about 40 °C (Dixon and Gonzales, 1994). Solid medium is poured into Petri dishes and left to solidify for about 20 minutes. For long periods of storage, Petri dishes with agar can be kept in a cool and dry place with stretch para-film sealing both top and bottom plates. To avoid contamination by evaporation and condensation, Petri dishes can be placed upside down. This way, the Petri dishes are ready for initiation of callus from the germination of the seeds and subsequent growth of callus cultures. On the other hand, to prepare liquid media, all the ingredients are added, except for the agar (Puad, 2011, Krishnan, 2009). Table 4-2 Supplements for the Murashige and Skoog (MS) media. Sulfur free MS (SFM) additionally was supplemented with 100 – 400ppm DBT.

Suspension liquid medium Solid seed germination Callus solid Medium type (The same as callus medium in 1 liter medium solid medium except agar)

MS or SFM MS 4.4 g L-1 (Encina et al., Sucrose 30 g L-1 2001) -1 MS 4.4 g L-1 NAA 1 mg L -1 MS 4.4 g L-1 Sucrose 30 g L-1 IAA 1 mg L -1 Sucrose 30 g L-1 NAA 1 mg L-1 2,4-D 0.5 mg L -1 Myo-inositol 0.2 g L-1 IAA 1 mg L-1 BAP 0.5 mg L

Agar 8 g L-1 2,4-D 0.5 mg L-1

BAP 0.5 mg L-1 4.4 g L-1 MS Agar 8 g L-1 (May and 30 g L-1 sucrose Leaver, 1993) 0.5 mg L-1 NAA 0.05 mg L-1 kinetin

Prior to the start of the experiments, it is essential to prepare stock solutions which can then be ready for use anytime during the laboratory work, as well as other equipment such as; • Chemical balance, with accuracy of 0.0001 g, weighing capacity of 120g. For accurate weight for the chemicals and the cells. • Hanna pH meter, range -2.00 to16.00 pH, accuracy ±0.01 (excluding probe error) to adjust the medium ph. • Rodwell series 32 Autoclave floor standing top loading to sterilize the medium and the equipment. • Airstream Vertical Laminar Flow Clean Benches (laminar flow cabinet) to work under sterile conditions. • Open platform orbital shakers, in a constant temperature room at 25 ˚C with fluorescent lighting (Dixon and Gonzales, 1994). Other sources experimental errors due to measurement accuracies can be listed as: • Measuring cylinder with accuracy of 0.1 ml • Spectrophotometer (Shimadzu uvmini-1240 uv-vis spectrophotometer) • Gilson pipettes (All pipettes used were calibrated annual external by STARLAB (https://www.starlabgroup.com).However, the accuracy of the pipettes was monitored regularly in the lab by weighing different amount of water (Density of water is 1 g/ml).

65 4.2.1. Preparation of Essential Compounds for Medium All the chemicals, which were used for the preparation of the stock solutions, were bought from Sigma Aldrich, UK (Sigma-Aldrich.) . 4.2.1.1 Preparation of sodium hypochlorite This solution is used to sterilize the explants and the seeds. The concentration of the chemical was 4% v/v, therefore, to make a concentration of 1 % v/v of this chemical; 37.5 ml of sodium hypochlorite was dissolved in 112.5 ml of water. 4.2.1.2. Preparation of 6-Benzyl Amino Purine (6, BAP) The stock solution was prepared by adding 0.00225g of 6, BAP into 100ml of distilled water. Finally, by syringe filtration method the solution can be sterilized and then kept at 4°C. 4.2.1.3. Preparation of 2, 4-Dichloro phenoxy acetic acid (2, 4 D) The stock solution was prepared by adding 0.0221g of 2, 4-D into 100ml of distilled water. Finally, by syringe filtration method the solution was sterilized and then kept 4°C.

4.2.2. Preparation of Essential Components for Chemical Analyses All chemicals used in this study were analytical grade and were procured from sigma, UK (Sigma-Aldrich.). 4.2.2.1. Preparation of DBT Due to the insolubility of DBT in water, an organic solvent was chosen according to its solubility. The organic solvent chosen was ethanol; because DBT is soluble in ethanol at a ratio of 1:100. Also at low concentration ethanol is harmless to plant cells and sometimes they may even produce ethanol themselves (Mavituna, personal communication). 4.2.2.2. Preparation of 2-Hydroxy benzyl phenyl (2- HBP) The 2-HBP was prepared by dissolving 250mg in 100ml of absolute ethanol. The concentration of the solution was 2.5 g/l and this solution was used as the standard solution in DBT analysis. 4.2.2.3. Preparation of Gibb’s assay To prepare Gibb’s solution10 mg of Gibb’s reagent (2, 6-dicholoroquinone-4-chloroimide) was dissolved in 10 ml ethanol (Yaqoub, 2012). 4.2.2.4. Preparation of fluorescein diacetate (FDA) The FDA stock solution was prepared in acetone by dissolving 20 mg of FDA in 10 ml acetone and stored in a refrigerator at 2-5 OC. Before adding the stock solution onto the cells, it was diluted by adding distilled water (Widholm, 1972) and (Dixon and Gonzales, 1994).

66 4.2.3. Preparing Saline Solution Saline solution was prepared by diluting 9g of sodium chloride in a 1 litre Duran bottle, then autoclaved before being used.

4.2.4. Preparation of pH Adjustment Chemicals The pH level in the medium was balanced by adding sodium hydroxide as a base to raise the pH level and hydrochloric acid to decrease the pH.

4.3. Plants, Callus, Cell Suspension and Hairy Root Cultures

4.3.1. Initiation of Cell Suspension Cultures Starting from the Seeds 4.3.1.1. Sterilization and germination of seeds The seeds of Arabidopsis thaliana (thale cress, or mouse-ear cress) and Nicotiana tabacum (Tobacco) were obtained from the Faculty of Life Sciences, the University of Manchester. As for the horseradish (Armoracia rusticana), fresh roots with growing points were carefully chosen from a local supermarket (Tesco) and grown in the soil. Firstly, all seeds were sterilized before germination. Arabidopsis thaliana seeds are very small; as shown in the results in Section Chapter 0Therefore, 10 to 20 seeds were soaked carefully in absolute ethanol for 1 minute and then transferred to 1% (v/v) sodium hypochlorite in a small beaker for 20 minutes. In the final step, the seeds were transferred into another beaker containing sterile distilled water for the rinsing step (3-5 times). The same steps can be used to sterilize other seeds and the leaves of horseradish (Negrutiu et al., 1975). The sterilized seeds of Arabidopsis thaliana and Nicotiana tabacum were transferred into seed germination MS medium in Petri dishes. The medium should be prepared prior to the sterilization step using the compositions in Table 4-2. After sterilization in the autoclave, the medium is poured into Petri dishes or small bottles and left to solidify for about 20 minutes. The petri dish or the bottle should be closed carefully and para film can be used to seal them to avoid any contamination. Finally, the seeds are placed under light in the temperature controlled room (25oC) (Dixon and Gonzales, 1994).

4.3.1.2. Callus initiation and maintenance from leaves and shoots • MS medium was used for callus initiation for all the plants as shown in Table 4-2.

• After 2-3 weeks of the seeds germination, leaves and shoots appeared and were harvested as shown in Section 6.1. Results of Molecular Docking for Biodesulfurization Enzymes

67 • Leaves and shoots were cut into smaller pieces to increase the surface area under aseptic conditions before being transferred into callus medium; using a sterilized scalpel.

• The same step was done for horseradish leaves, which were placed into callus medium immediately after sterilization. There is no need for the seeds germination step as mentioned in Section 4.3.1.1. Sterilization and germination of seeds

• Callus must be ready for the first subculture after four weeks.

• Callus must be sub-cultured every three weeks, the callus colour will change to yellow after many subcultures as shown Figure 6-11 (Mavituna et al., 1988, Krishnan, 2009, Puad, 2011).

4.3.1.3. Initiation and maintenance of cell suspension cultures The fresh medium was prepared by dissolving all the components in distilled water as shown in Table 4-2 and the pH was adjusted to 5.8. The liquid medium was sterilized in the autoclave and then left to cool at room temperature. When transferring 10ml cells from suspension cultures into 250ml sterile Erlenmeyer flasks containing 90ml fresh medium, it is recommended to open and close the flask quickly and the nick of the flask can be flamed before closing it to avoid any contamination. Flasks were incubated on an orbital shaker at 110rpm and 25 ˚C. After 2 to 3 weeks, the sub-culturing was complete, and bulking up of the cell cultures commenced.

On the other hand, solid medium is considered as long-term maintenance, longer than suspension cultures. Therefore, seed germination is used to obtain callus, which can be maintained.

• 50ml of cell suspension liquid medium (MS or SFM) was poured under aseptic conditions into 125ml sterilized Erlenmeyer flasks. These flasks were then inoculated with about 1g of callus aggregates and sealed again with the cotton bung and aluminium foil to avoid contamination.

• The flasks were then placed on an orbital shaker at a speed of 110rpm at 25± 1°C with continuous illumination of 2000 Lux.

• Suspension cultures were sub-cultured under sterile conditions every 3 weeks in the

68 first 3 months. After this, the sub-culturing was every 10 days.

• During incubation, the callus and suspension cultures were maintained, and continuously growing and bulking up. This was done to avoid losing cells and starting the process again from the beginning.

4.3.2. Initiation of Suspension Cells from the Explants Grown in Soil Some plant seeds and roots (such as horseradish) can be germinated and/or grown in either normal compost or in sterilized compost. Once healthy leaves and shoots are obtained, these can be used as explants to initiate callus in MS medium. The following procedure can be used:

i) Firstly, spraying the soil with some water to moisten it before sowing the seeds. ii) After the soil becomes moist, the seeds are scattered evenly onto the surface of the soil and left until the leaves and shoots appear. iii) The leaves and shoots are harvested and then surface sterilized before transferring into callus induction medium. iv) For the surface sterilization, leaf and shoot explants are washed well with tap water and then soaked in 70% (v/v) ethanol for one minute. After that, they are transferred into 5% (v/v) sodium hypochlorite for 10 minutes (Negrutiu et al., 1975). v) The explant was then washed around three times with sterilized distilled water to remove any leftover solution (Dixon and Gonzales, 1994). The steps following sterilization can be seen in Section 6.1 from the whole plant to the callus and finally the suspension cultures.

Now the explant is ready to transfer into the callus medium. Arabidopsis thaliana leaves are too small, so there is no need to cut them into smaller pieces. On the other hand, Nicotiana tabacum and horseradish leaves and stems are large and they need to be cut into smaller explants. They are transferred using sterilized forceps and pressed carefully into the agar. Once the calli are initiated, they can then be transferred to callus growth medium as explained previously.

4.3.3. Cell Culture Sieving and Maintenance As mentioned in Section 3.2. Plant Cell Culture Nicotiana tabacum produces highly- aggregated cell suspension cultures (Hashimoto and Azechi, 1988), (Hooker et al., 1990). This phenomenon would cause difficulty in obtaining homogeneous and reproducible samples as well as inocula. Therefore, cell sieving was the best technique to obtain nice and fine cell aggregates. A stainless steel sieve (Error! Reference source not found.) with hole s ize of 1- 2 mm was used to obtain nice and fine cell aggregates. The sieving size can affect the cell growth by reducing the number of viable cells as it may kill the cells when the small diameter is used by pressing the cells too much. Beforehand, all apparatus, metal sieves, several glass beakers, a glass bar, forceps, pipette tips and sufficient distilled water should be sterilized.

Figure 4-2 Metal sieves where the cells should be poured and use the glass bar to press the cells gently to the baker to obtain nice and fine cell aggregates. Under the laminar air flow the sieving was started by placing the metal sieve on top of the baker as shown on Figure 4-2 above. After shaking, the cell suspension was poured into the beaker through the sieve. The big aggregated cells remained in the sieves. Thereafter, the glass bar was used to spread them and allow the fin cells to go through the sieves. The sterile distilled water or medium helps by rinsing the cells two to three times. Finally, the sieved cells remain in the baker. The fresh suspension medium was then poured into the beaker and shaken well and then transferred to a new sterile shake flask and placed on the orbital shaker shaking at 120rpm. The fine cell suspension should culture for three to four weeks before any batch experiments to remove the dead cells and rescue living cells is initiated.

4.3.4. Contamination Test At the initial stage of the culturing, to avoid any contamination and loss of the plant cells, a test was performed only for the suspicious-looking flasks that were cloudy. Two types of agar media were prepared: Nutrient Agar (NA) and Yeast Extract Broth agar (YEB), the

70 composition of the latter given in Table 4-4. The Nutrient Agar was used for testing bacteria in the culture and YEB agar was used for testing yeast in the culture. The Nutrient Agar (NA) was prepared by dissolving Beef Extract 3.0g, Peptone 5.0g, and Agar 15.0g in 1L of distilled water with pH adjusted to 6.8. All media were sterilized by autoclaving at 121°C for 20 minutes. After sterilization, under aseptic conditions, the agar solutions were poured into Petri dishes at 30ml for each 10cm Petri dish and the plates were kept in stationary position for solidification. After solidification, a small amount of culture was spread by sterile rod into the agar plates. It was then incubated at 30°C for 24 to 48 hours. After the incubation period, the plates were checked for the presence or absence of yeast or bacteria.

4.4. Experimental Preparation Procedures Before any batch experiments are performed, preparation is the most important step. All the results depend on this step. To start any batch initiation all apparatus was autoclaved in advance, which included a 5ml measuring cylinder, beakers and the 100ml shake flasks (or Duran bottles). All the batches were initiated under a laminar air flow cabinet to ensure sterile conditions. Moreover, all experiments in aqueous samples were initiated using flasks stoppered with cotton and aluminium foil. However, for the experiments with crude oil, the cotton was replaced with a rubber bung and wrapped with Para-film following the required safety procedures in order to ensure the Nicotiana tabacum cells were kept under airtight conditions to avoid any crude oil vapour leaking into the atmosphere which is a breathing hazard and in large quantities, a fire hazard (Figure 4-3).

Figure 4-3 Flasks stoppered with cotton and aluminium foil and for crude oil experiments replace the cotton with rubber bung and wrap it with Para-film.

First, the standardization of the inoculum was prepared in a 500ml flask, by collecting all the cell suspension cultures in fresh medium, and leaving them on the shaker for 5-6 days to reach the exponential phase, before inoculation of the small flasks. A quantity of 20ml of fresh medium from the 500ml flask was distributed into 50ml samples flasks containing 10% of each sample inoculum. The technique for preparing the samples was done with care to make all samples as equal as possible with no contamination until the last sample was taken. The next process was the preparation of the samples and the distribution of the cells and the medium into small flasks. The inoculum of 500ml shake flask was taken with its opening flamed and the opening of the sterilized measuring cylinder ( Figure 4-4) was flamed to get 2ml of Nicotiana tabacum cells. An amount equalling 10% of the sample was poured into the flask; ensuring that no liquid remained in the cylinder. The 50/100ml shake flasks opening was then flamed and the cells poured into it. Using a syringe, 20ml of this media was taken and poured into the 50/100ml shake flask, including the Nicotiana tabacum cells. The opening of the flask was flamed and then secured well with either rubber or cotton. This process was repeated for the rest of flasks in every batch. After preparing the samples, all flasks for each batch were placed on the shaker at 120rpm under a constant light source with an illumination of 2000 Lux and room temperature of 26 °C.

Figure 4-4 5ml sterilized measuring cylinder For the experiments containing crude oil, the crude oil was added to samples by using a 5ml syringe under a fume cupboard and were then stoppered using their rubber bungs and aluminium foil and Para-film to prevent any possible evaporation of crude oil into the atmosphere Figure 4-5 . Moreover, as can be seen in Figure 4-5, the crude oil is not mixed well with the medium but a separate layer is formed. However, all the flasks were placed onto the shaker trying to allow the cells to contact the crude oil. The crude oil could not be

72 autoclaved due to the added hazard it presents at high temperatures nor could it be filter syringed using a syringe filter with a diameter of 22mm and 0.45 µm pore size because of its high viscosity. Thus, it was used in its unsterilized form. Since the use of crude oil necessitated the air-tight containers for culturing, (e.g. Duran bottles or flasks with rubber bungs), before the crude oil addition, as a control experiment, the cells were grown in the same media and at the same conditions without crude oil in airtight bottles. These control experiments with and without DBT were designed to investigate whether the air tight nature of the bottle, that does not allow for vapours of crude oil to leave the bottle, nor does it allow for oxygen transfer into the bottle, would affect the growth of the cells Figure 4-6.

Figure 4-5The picture shows 2ml Nicotiana tabacum cells and 2ml crude oil in 20ml of medium in airtight bottles for the batch experiments.

Figure 4-6The picture Shows 2 ml Nicotiana tabacum cells in 20 ml of MS medium in Duran bottles.

73 4.4.1 Incubation of Nicotiana tabacum Cells into Different Growth Media In all experiments after bulking up the cell suspension culture and obtaining enough cells, if needed, the cells should be sieved. The running of the experiments was started in a different growth medium. Each experiment was done in parallel. The experiments were done in different media and the biodesulfurization batches were supplemented with DBT in the absence or in presence of crude oil. Most of the batch experiments were run for three or four weeks. Two flasks were sacrificed for taking a sample for the growth measurement every three to four days in order to increase the reliability of the results obtained. Some of the experiments were repeated more than once to get an accurate result. The details of all batch experiments operated with Nicotiana tabacum cells cultures in different types of media and different conditions are tabulated in Table 4-3.

Table 4-3 Batch experiments operated with Nicotiana tabacum cells cultures in different types of media and different conditions. These media codes* are used later in Chapter 7

Media Air transfer (oxygen Air-tight (no oxygen DBT Crude oil Code* transfer) transfer)

MS ✓ MS.D ✓ MS100 ✓ ✓ MS200 MS.DO.100 ✓ ✓ ✓ SF.100 ✓ ✓ SF.D.100 ✓ ✓ SF.100.D.O. SF.200.D.O. ✓ ✓ ✓ SF.300.D.O. SF.400.D.O. *Codes: MS= Murashige and Skoog basal medium. SFM= Sulfur-free Murashige and Skoog basal medium. D= Duran bottle (No air exchange). The code without D means in normal shake flask with cotton bung allowing air exchange. O= crude oil (2 ml in 20 ml medium). 100/200/300/400= DBT concentration in ppm.

74 4.5 Analytical Techniques For the sample analysis of the chemical compounds, as well as measurement of fresh and dry cell weights, the following equipment and techniques have been used: a digital balance, vacuum filtration, High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC).

4.5.1. Fresh and Dry Weight Measurements The growth rate of Nicotiana tabacum or any other plant such as Arabidopsis thaliana can be monitored by taking fresh and dry weight measurements of the culture samples. After many sub-culturings, experiments were performed over three weeks in batch cultures.

After recording the sample volume, the cells were separated from the supernatant liquid by filtering using the Büchi vacuum pump V-500 as shown in Figure 4-7. The supernatant was stored in 12ml centrifuge tubes at -30˚C for further analysis e.g. sucrose or DBT analysis. The fresh cells were collected on Whatman filter paper with a pore size of 0.2µm with polypropylene funnel. For fresh and dry weight measurement, the dry and wet filter paper weights were recorded before being used for cell filtration and collection. The filter paper without the cells was placed into an 80 °C oven for an hour to remove any moisture within it. The dry weight of the paper was then recorded. Then, this filter paper was wetted with distilled water and excess water filtered and wet filter paper weight was recorded. The wet filter paper with the fresh cells were weighed and the new weight of cells in the sample was calculated by the difference between these two “wet values”. To determine the dry weight, the same filter paper with the cells was kept in a drying oven at 80 ˚C for 48 hours after the fresh weight measurement. Then, the cell dry weight was calculated by subtracting the weight of the dry filter paper from the weight of the dry filter paper with the cells.

Figure 4-7 The vacuum filter to separate the cells from the medium in the culture samples.

75 The fine details of these procedures are as follows: the wet filter paper was put into the Buchner funnel. This was done by first laying the filter paper on top of the funnel and pressing down with metal forceps. The funnel was placed through a rubber bung which was placed into the Buchner flask. The vacuum was turned on and the sample poured into the filter and left for five to seven minutes. The vacuum drew the filtrate into the flask, leaving the cells on the filter paper. The forceps were used to take the wet paper with fresh cells out of the Buchner funnel and onto some aluminium foil. The filter paper with the new weight of the cells was weighed and placed into an oven at 80oC to dry for 48 hours to obtain the dry weight of the cells Figure 4-8. Filtrate was poured into 12ml centrifuge tubes and frozen ready to be syringe filtered into analysis bottles for sugar, DBT and 2-HBP analysis. In the experiments which contained crude oil, the same procedure was followed however, first, the cells were allowed to precipitate and crude oil to separate from the aqueous medium. Then, the crude oil was pipetted out by carefully using a 5ml pipette. The pipetted crude oil always contained a small amount of aqueous medium which is separated as will explain in Section 4.5.2. Sample Preparation for GC-FID and HPLC Analysis Due to crude oil’s potential hazards, the filtration, the weighing and wetting of filter paper took place within the fume cupboard.

The dry and fresh weight concentrations were calculated as follows:

(Dry⁡weight⁡of⁡cells⁡&⁡filter⁡paper⁡(g)−Dry⁡weight⁡of⁡filter⁡paper⁡(g)) x⁡1000ml⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡Equation⁡4-1 Total⁡amount⁡of⁡the⁡sample⁡(ml) (Fresh⁡weight⁡of⁡cells⁡&⁡filter⁡paper⁡(g)−Fry⁡weight⁡of⁡filter⁡paper⁡(g)) Equation 4-2 x⁡1000ml⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡ Total⁡amount⁡of⁡the⁡sample⁡(ml)

Figure 4-8 Fresh and dry cells on Whatman filter papers.

76 4.5.2. Sample Preparation for GC-FID and HPLC Analysis The aqueous samples were analysed for their sucrose, glucose and fructose concentrations in the HPLC. Aqueous and crude oil samples were analysed for their DBT and 2-HBP as well as some sulfur compounds such as 4,6-DEDBT (4,6-Diethyldibenzothiophen) 4-MDBT (Miethyldibenzothiophen) concentrations in the GC-FID. To prepare the samples for the GC- FID and HPLC analyses, the frozen filtrates from samples were first defrosted and syringe- filtered into 2ml sample vials Figure 4-12. A 22mm diameter and 0.45µm pore size cellulose nitrate membrane syringe filter Figure 4-9 was used to filter the samples from any cells remaining in the supernatants. There was no need for a sterilized one; only a new-clean syringe should be used for each sample to avoid cross contamination.

Figure 4-9 A 22mm diameter and 0.45 µm cellulose nitrate membrane syringe filter to analyse the sugar and sulfur components in the free cells media by HPLC and GC-FID .

The crude oil samples that contained a small amount of aqueous medium were centrifuged for 10 minutes at 10000rpm to separate and remove the aqueous part Figure 4-10and Figure 4-11 (Yu, 2006). Finally, under the fume cupboard liquid and crude oil separation, they were injected into the samples vials. There were 2 vials from each sample: the liquid vial Figure 4-12 for HPLC and GC-FID analysis and the crude oil for GC-FID.

Figure 4-10 Crude oil was mixed with the medium; it was difficult to inject the crude oil and the medium separately in the small vial.

Figure 4-11 The crude oil and the medium were separated by using a centrifuge 10000rpm for 10 minutes.

Figure 4-12The crude oil and the medium in separate vial ready for GC-FID and HPLC analysis.

78 4.5.3. Sugar Analysis by High Performance Liquid Chromatography (HPLC) Plant cells in culture hydrolyse sucrose were analysed by converting them into glucose, and fructose into the medium before taking up glucose and fructose from the medium. Sucrose analysis should, therefore, be accompanied with glucose and fructose analyses. In section Error! Reference source not found., the supernatant was collected after filtering the s amples and stored in centrifuge tubes at - 30˚C. The supernatant can be used to measure the concentration of the sugars and the standards samples of sucrose, glucose and fructose by high performance liquid chromatography (HPLC). The stock solutions of the sugar standards were prepared at concentrations of 35g/l sugar in distilled water. Then the solutions were sterilized using syringe filtration (Pore size: 0.45µm) and then they were used as the stock solutions for the preparation of standards. The stock solutions of (sucrose, glucose and fructose) were diluted to 17.5, 8.75, 4.375, and 2.18 g /l.

The High-Performance Liquid Chromatography (HPLC) (Varian (USA)) equipment was fitted with the PL Hi-Plex Ca column (300 × 7.7 mm) (Polymer Labs) and a disposable guard cartridge (Polymer Labs) was used for the sugar analysis. The mobile phase was HPLC grade water and it was passed through the column at a flow rate of 0.4ml min-1. The analyser was equipped with an Evaporative Light Scattering Detector (ELSD, Polymer Laboratories, and UK) and a computerized integrator (Prostar/ Dynamax System).

Calibration curve for known concentration of sucrose, glucose and fructose should be plotted to calculate the sugar consumption in the samples during the cells growth

Figure 4-13. Each experimental standard should be run in HPLC before the samples; to get accurate results. The figures below give an example of one of the sugars calibration curves. To double check retention time by preparing mixture of the 3 sugars with 30g/l see in Figure 4-14 the HLPC chromatogram of sugar (sucrose, glucose, and fructose) mixture. In order for BDS using plant cell and tissue culture to become a reality more researched is needed. It can be speculated that with intensive and interdisciplinary research plant-based biocatalysis for BDS can be used as complementary stage for finishing of desulfurization of crude oil.

180 y = 4.624x + 0.7146 160 Fructose 140 y = 4.4897x + 6.0157 120 Glucose 100

80 Peak area Peak 60

40 y = 2.1907x + 1.564 20 Sucrose 0 0 5 10 15 20 25 30 35 40 Conc. g/l

Sucrose g/l Glucose g/l Fructose g/l

Figure 4-13 Calibration curve for sugar analysis.

Figure 4-14 HLPC chromatogram of sugar (sucrose, glucose, and fructose) mixture (area against time), retention time of sucrose, glucose, and fructose were 11 minutes; 12.968 minutes; and 15.803 minutes respectively.

80 4.5.4. DBT and 2HBP Analysis

To determine the degradation of Dibenzothiophene (DBT) and the appearance of sulfur components by the biodesulfurization process in Nicotiana tabacum, two instruments has been used: the Spectrophotometer UV and Gas chromatography (GC/FID).

4.5.4.1. Gibss’ assay Using UV Spectrophotometer The Nicotiana tabacum ability to desulfurizing DBT (as S-source only) was determined using Gibb's assay, (2,6-dicholoroquinone-4-choroimide). In this assay, 2,6-dicholoroquinone-4- choroimide reacts with 2-hydroxybiphenyl. The result is 2,6-dicholorobenzenoneindophenol of which gives an intense blue colour to the solution which can be measured at 610nm. The blue colour shows that DBT was transformed to 2-HBP and sulfur removal was successfully achieved (Ibrahim, 2017, Papizadeh et al., 2010). Moreover, Gibb's assay is not only for detecting 2-HBP but is also used for the detection of other phenol derivatives (Gibbs, 1926). From the cell-free supernatants, the amount of the accumulated 2-HBP (the end products of the biodesulfurization of DBT via 4S pathway) can be determined from the standard curves by preparing different known concentration of 2-HBP. Firstly, the pH of the standards was adjusted to 8.00. Secondly, 10μl of Gibb's reagent was added to 1ml of 2-HBP solution. Subsequently, the standards were kept at room temperature for 30 minutes Figure 4-15 and Figure 4-16 to develop the coloured complex. Finally, the intensity of the blue colour was measured at 610nm by using Spectrophotometer UV for optical density measurement which can be plotted against the different concentration of 2-HBP. From standard, the unknown concentration of 2-HBP in the samples can be calculated Figure 4-17 (Gupta et al., 2005); (Hussein Al-Jailawi, 2015).

Figure 4-15 Blue colour with Gibbs’ reagent indicating the presence of 2-HBP at different concentration of the standard.

Figure 4-16 The 2-HBP standards at zero minutes on the left and after half an hour on the right.

450 400 350 300 250 200

HBP Con.HBP (ppm) 150 -

2 y = 356.83x 100 50 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 OD (610 nm)

Figure 4-17Calibration Curve for 2-HBP.

82 4.5.4.2. Gas Chromatography-Flame Ionization Detector (GC-FID) Analysis A gas chromatograph equipped with a flame ionization detector (FID) (HP-6890 GC/ FID) was used for the analysis of DBT and 2-HBP in the samples. The column used for the aqueous samples was J&W Scientific DB-5MS which was a fused silica capillary column (30 meters long) (250μm x 0.10μm nominal). The equipment was supplemented with helium carrier gas at a flow rate of 1.5ml min-1, hydrogen at 50ml min-1, and air at 450ml min-1 with helium make up flow at 30ml min-1. The GC/ FID oven was programmed from 80° C to 250° C with an initial hold time of four minutes and a ramp rate of 8° C min-1 and a final hold at 250° C for 1.0 minute. The sample volume injected was 1.0 or 3.0μl. The samples were analysed in duplicate and from the analysis the amount of DBT in the sample could be determined using the calibration curves(Wauquier, 1995);(Wardencki, 2000) (Yaqoub, 2012) For the crude oil samples, after separating them from aqueous medium, a different column was used for the analysis of DBT and sulfur compounds. The column used was HP-1(Model No: Agilent 19091Z-102) Max 325° C and the column was a Methyl Siloxane column (25 meters long) (200μm x 0.33μm nominal). The equipment was supplemented with helium carrier gas at a flow-rate of 1.5ml min-1, hydrogen at 50ml min-1, and air at 450ml min-1 with helium make up flow at 30ml min-1. The GC/ FID oven was programmed from 40° C to 285° C with an initial hold time of 15 minutes and a ramp rate of 8° C min-1 and a final hold at 250° C for 1.0 minute. The column information and GC-FID setting up was from (http://www.restek.com). The sample volume injected was 2.0μl, because the remaining crude oil was a very little amount (Wauquier, 1995). First of all, the calibration curve for known concentration in ppm should be plotted to calculate the unknown concentrations of DBT and 2-HBP and other sulfur compounds (such as 4, 6 DEDBT, 4MDBT) in the samples. For each use of the GC-FID for a set of samples, the standards should be run before the samples to get accurate results. To obtain the calibration, the standards of these sulfur compounds should be prepared in different concentrations. However, these compounds are insoluble in water, so absolute ethanol was the chosen as the organic solvent because they are soluble in ethanol at a ratio of 1:100 (Yaqoub, 2012). The standards for DBT and the other compounds 2-HBP, 4,6 DEDBT, 4MDBT were prepared by using each compound in absolute ethanol at 10000, 5000, 1000, 100 and 10ppm. Using these standards in ethanol, additional standards in fresh medium (without cells) and in distilled water were prepared at 150ppm, 100ppm, 80ppm, 60ppm, 10ppm. All these standards in absolute ethanol, fresh medium and distilled water, were run in DB-5MS, fused

83 silica capillary column. The calibration curves for the standards of DBT and 2-HBP in absolute ethanol, medium and distilled water are shown in Figure 4-18,4-19 and 4-20. Additionally, the standards in ethanol were run again in HP-1, Methyl Siloxane column which was to be used for the analysis of sulfur compounds in crude oil Figure 4-21. To distinguish between ethanol peak and other compounds peaks, pure absolute ethanol run and appear at 1.1 minutes as seen in Figure 4-22 and 4-23 shows the 200ppm standards of DBT in ethanol, fresh medium and distilled water eluting from the DB-5MS, fused silica capillary column, ethanol peaks in part A and DBT peaks in part B. Similarly, Figure 4-24 shows the elution of 200ppm 2-HBP standards.

14000 14000 12000 12000 y = 1.2912x y = 1.1842x R² = 0.9944 10000 R² = 0.9599 10000 8000 8000

Area 6000 Area 6000 4000 4000

2000 2000 0 0 0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000 DBT Con. ppm 2-HBP Con. ppm

Figure 4-18 Calibration curve of DBT dissolved in absolute ethanol in the left, and 2-HBP dissolved in absolute ethanol in the right.

3000 400 y = 18.452x 350 y = 2.3328x 2500 R² = 0.9735 300 R² = 0.9015 2000 250

1500 200

Area Area 1000 150 100 500 50 0 0 0 50 100 150 200 0 50 100 150 200 DBT con. ppm 2-HBP con. ppm

Figure 4-19 Calibration curve of DBT dissolve in medium in the left, and 2-HBP dissolve in medium in the right.

2500 450 y = 2.6114x 400 R² = 0.8303 2000 350 y = 13.715x 300 1500 R² = 0.9956

250 Area 1000 Area 200 150 500 100 50

0 0 0 50 100 150 200 0 50 100 150 200 DBT con. ppm 2-HBP con. ppm

Figure 4-20 Calibration curve of DBT dissolve in distilled water in the left, and 2-HBP dissolve in distilled water in the right.

5 180 4.5 y = 0.0112x + 1.4939 160 y = 0.5677x + 35.199 4 R² = 0.8313 140 R² = 0.8554 3.5 120 3 100 2.5

Area 80 Area Area 2 60 1.5 40 1 0.5 20 0 0 0 100 200 300 0 50 100 150 200 250 2-HBP con. ppm DBT Con. ppm

Figure 4-21 Calibration curve from running the standards in HP-1, Methyl Siloxane column, DBT dissolve in medium in the left and 2-HBP dissolve in medium in the right.

Figure 4-22 Ethanol peak at retention time 1.1 minute in pure ethanol and ethanol in fresh medium elute from the DB-5MS.

86 (A)

(B)

Figure 4-23 Calibration curves for 200ppm of DBT standards which were prepared in different solution in the DB-5MS column (area against time). A- Shows ethanol peaks at 1.1, B-Shows DBT peaks at 17 min in fresh , ethanol and no peak in distilled water at 17 min .

(A)

(B)

Figure 4-24 Calibration curves for 200ppm of 2-HBP standards which were prepared in different solution in the DB-5MS column (area against time) A-Shows ethanol peaks at 1.1, B-Shows 2-HBP peaks at around 14 min in fresh medium, ethanol and distilled water.

88 After several calibration trials in the DB-5MS, fused silica capillary column, the calibration method chosen involved the use DBT and 2-HBP, separately, dissolved in absolute ethanol at a ratio of 1:100 which were then used to prepare DBT and 2-HBP standards at various ppm levels in fresh aqueous medium. Moreover, increasing the injection volume from 1 to 3 µl had a positive impact on the results.

On the other hand, standards of DBT, 2-HBP and other compounds (4,6-DEDBT and 4MDBT) were run in HP-1 column to check the retention time. The area for DBT was: 35.5 at 1.5 retention time and 261 for 2-HBP at 1.9 retention time and other compounds seen in Figure 4-25. The retention time illustrated in two standards of DBT at 1.5 minutes and 2-HBP at 1.9 minutes at 200ppm concentration were run as shown in Figure 4-26.Pure crude oil was run in the HP-1 column to detect DBT, 2-HBP and other compounds (4,6-DEDBT and 4MDBT) in the same time as in the standards as seen in Figure 4-27.

300

250 261 200

150

100

50 73 35.5 1.4 0 DBT at 1.5 min 4,6-DEDBT at 1.6 min 4MDBT at 1.7 min 2-HBP at 1.9 min

DBT=Dibenzothiophene/ 4,6-DEDBT=4,6-Diethyldibenzothiophen/ 4MDBT=Miethyldibenzothiophen/ 2-HPB=2-Hydroxyblphen

Figure 4-25 Some Sulfur compounds area in pure Crude oil

Figure 4-26 200ppm standards for DBT peak at 1.5 and 2-HBP peak at 1.9 at 100:1 ratio (area against time).

Figure 4-27 The pure crude oil peaks in the HP-1 column (area against time).

4.6. Cell Viability Tests Studying biodesulfrization in presence of crude oil was started from cell viability test to check whether the plant can survive after contacting the crude oil or not. The crude oil which was used in this study was obtained from Stanlow Oil Refinery (Yaqoub, 2012).

In this study, washing techniques are used to determine cell viability and predict the effect of bringing plant cells into contact with crude oil. However, this is a novel process and literature surrounding this area is still limited. It has been shown, in the literature, the effect of oil when it comes into contact with plants. In other words there is no strong indication on how disruptive oil is (Boniek et al., 2015). However, through these experiment results it is possible to examine the effects of oil spillages on plant life. Also, similar behaviour in plant cells can be predicted. Therefore, the effects of oil on whole plants can be studied (Setti et al., 1996). Moreover, to prevent any potential disruption to the cell, the cell washing technique should be developed. Because it is important to remove all traces of crude oil from the surface of the Nicotiana tabacum cells during the experiments. Moreover, it must be taken into account that washing the cells after contact with oil would not wash away the internal part of the cell, as the oil may penetrate the cells and affect them internally. Therefore, cell viability tests can be used to detect if there is any cell damage which should be taken into consideration.

4.6.1 Removing Crude Oil by Washing with Saline Technique In order to prevent osmotic shock of plant cells were washed with saline solution instead of distilled water. The washing process took place in the fume cupboard where the crude oil should be used. This was because this work cannot be done in the laminar air flow cabinet due to safety regulations. It was important to work fast and carefully to avoid any contamination. Moreover, all procedures done in the laboratory were conducted with personal protective equipment worn; a laboratory coat, nitrile gloves, and goggles. Washing technique was using for both experiments, cell viability test by using fluorescein diacetate and cell viability experiment by observing growth.

92 4.6.2 Cell Viability Test by Using Fluorescein Diacetate

The fluorescein diacetate staining is a method used for checking the plant cell viability. The plant esterases enzyme in the cell causes the following reaction Figure 4-28 in which produces dissociated fluoresces green under the UV microscope.

Figure 4-28 Reaction scheme of fluorescein diacetate hydrolysis catalyzed by plant esterases (PE) (Vitecek, 2007 et al.,)

The sub-cultured callus cultures that would have been left to grow will be separated equally into Duran bottles using sterilized plastic forceps. In each of the Duran bottles there is crude oil under a fume cupboard. The samples were of two types:

1) The cells immerse in medium with crude oil and leave it for (0h, 30min, 1h; 2hrs; 1d; 8days; and 12days). 2. The cells immerse in crude oil only (0h; 1h; 12 days).

Initially the sticking of crude oil was noticeable in the cells with free medium crude oil and crude oil with medium after washing the cells as shown in Figure 4-29.

Figure 4-29 A- Fresh and dried cells after contacting free medium crude oil, which show the sticking of crude oil on the cells. B- Fresh and dried cells after contacting crude oil with medium which show less crude oil sticking on the cells. The samples are washed using a sterile saline solution (0.1 %). It is used in order to overcome osmotic pressure. After the cells have been washed thoroughly by adding saline solution and rinsing out the Duran bottle several times, they are placed with FDA which was diluted in acetone (Yaqoub, 2012) and left for 2 minutes and then placed on the slides to take the pictures (Dixon and Gonzales, 1994).

To test the cell viability, a stock solution of fluorescein diacetate (FDA) was prepared (4.2.2.4. Preparation of fluorescein diacetate (FDA). The FDA solution was diluted by adding distilled water, which will form a cloudy white suspension. On a microscope slide, this FDA stain is added to cells and given time for the stain to penetrate the cells. The microscope slide will be covered with a cover slip and will be viewed using an Ultraviolet fluorescent microscope with a blue/violet filter. Finally the viability of the cells can be seen by noticing the radiant cells with green colour. This means that they are still alive after being in contact with crude oil. Then pictures were taken under UV microscope with 490nm wavelength and 2 different colours (green light, overlying [black and white on the green]).

94 4.6.3. Monitoring Nicotiana tabacum Culture Growth Subsequent to Contacting with Crude Oil at Various Length of Times

The initiation procedure used for the BDS experiments was used for the cell viability growth experiments after leaving the cells in contact with crude oil for different times, before being washed with saline solution to get rid of the crude oil and then put the Nicotiana tabacum cells back to grow in MS medium. For this experiment, there was no need to detect the DBT concentration or consumption. The analysis involved only filtration; to determine the cell dry weight and use HPLC to calculate sugar uptake. The objective of this experiment was to check Nicotiana tabacum cells’ ability to grow after being in contact with crude oil and washed with a saline solution. This will give evidence into the Nicotiana tabacum cells’ capacity to be used as a catalyst, if the cells can grow after they have been put back in media, this will show that they are still viable and can possibly be re- used; which is something that would be ideal for a catalyst, especially in large scale industries (Widholm, 1972; Satyawali, 2014).

Nicotiana tabacum cells which are in contact with crude oil were added to the cells in airtight shake flasks for 2; 4; 6; 8; and 24 hours. Six samples were taken for each contact time; producing 30 samples in total. After the Nicotiana tabacum cells were put in contact with crude oil for the a aforementioned times, the cells were washed with saline solution to get rid of the remaining crude oil. Firstly, the Saline solution was added to the sample of crude oil and liquid subculture in the flasks, each sample was shaken a little and left for the cells in it to settle. Secondly, it was then carefully poured into waste bottles before the cotton and foil were carefully put back on top of the flask. Finally, after washing with saline solution, the clean cells were moved into the laminar air flow cabinet. Then the cells poured into new sterilized flasks containing 20ml of MS. The flasks which had once contained crude oil were not flamed to avoid any potential combustion occurring. The new flasks which contain MS medium only were flamed by using Bunsen burner and left to grow for 17 days. The last step involved taking samples every three days to analyse sugar uptake and dry weight growth.

95 4.7. Hairy Roots This experimental procedure involves investigating the potential of plant hairy root cultures’ potential to degrade dibenzothiophene (DBT), a model compound used to represent the sulfur compounds in crude oil. Firstly, the Arabidopsis thaliana and Nicotiana tabacum will be grown in small shake flasks (25-500ml) in sterilized MS without DBT; also will the Horseredish leaves and roots as well for hairy root initiation using Agrobacterium rhizogenes.

Experimental Procedures: 1- Acquirement of A. rhizogenes: The master culture of Agrobacterium rhizogenes strain 15834 was in a freeze-dried state and obtained from ATCC (American Type Culture Collection). The experiment started with opening a Single-Vial to revive Agrobacterium rhizogenes, so the cell suspension could be recovered from the glass ampoule. A- Firstly, the work was carried out in a laminar flow cabinet. B- Using a small, sterile sharp blade, the neck of the ampoule is lightly marked (Figure 4-30). C- The ampoule is sanitized by wet gauze with 70% ethanol. Then the gauze is folded around the ampoule and broken at the marked area. D- It was ensured that the gauze was handled with care, as it should not be too wet with ethanol to avoid sucking ethanol into the culture and destroying the bacteria when the ampule is opened (www.atcc.org).

2- Revive: The lyophilized bacterial culture should be revived. After opening the ampoule, some sterilized yeast extract broth (YEB or ATCC) medium is poured in (see the components in Table 4-4) and mixed up with bacteria. The bacteria then transferred into Petri dishes containing YEB or ATCC solid medium and incubated for 2-3 days Figure 4-31. All the work was done under sterile conditions in a laminar flow cabinet to avoid any contamination like the one shown in Figure 4-32 (wang, 2006).

3-Preparation of the bacteria for inoculation: Agrobacterium rhizogenes were inoculated in 20ml liquid medium (pH =7.2) on a shaker at 150rpm at 25-30 ˚C for 24 hours. The bacteria were then transferred into 50ml YEB or ATCC medium, and cultivated for a further 24 hours in order to obtain an adequate bacteria density above OD 600, which can be determined by a spectrophotometer Figure 4-33.

96 4-Sterilization of the plant: Nicotiana tabacum was grown in vitro in seed germination medium, so there was no need for the sterilization step, however, for Armoracia rusticana (Horseradish) explant was used which was sterilized as mentioned in section 4.3.2.

5- Transformation or induction of hairy roots: The leaves and roots were infected by making a wound and then placing it in Petri dishes filled with the liquid bacterial solution for one minute. The leaves were then transferred to 250ml flasks containing 60ml MS-free liquid medium (Vivanco, 1992) supplemented with 3 % (w/v) sucrose for 24 hours or infecting the leaves and roots using a fine needle to inject the bacteria suspension culture as shown on Figure 4-34.

6-Preparation of the component to eliminate the bacterium in the hairy root initiation experiment: preparing a solution of 250mg/l of Cefotaxime in sterilized distilled water.

7- Eliminate Agrobacterium rhizogenes: Cefotaxime added to the cultures and left for one to 24 hours in order to eliminate (kill) the bacterium.

8- Transfer to MS-free hormone medium alone: Only leaves that developed the hairy root phenotype were sub-cultivated. Cultivation was in MS-free hormone nutrient medium, supplemented in Erlenmeyer flasks (250ml) with 60ml medium on a shaker (110rpm) at 26°C and pH 5.8. (Kastell et al., 2013); (Flores et al., 1987), or in solid MS-free hormone medium (Vivanco, 1992).

The cultures were sub-cultivated every 2 weeks and used in experiments four months after establishment. These experiments were performed in contained and sealed sterilized vessels (shake flasks ranging from 25 to 500ml and a jar bioreactor of 1L volume). The experiments involved measuring the growth rate by fresh and dry weight, and the culture samples were analysed for sucrose, glucose, fructose (by HPLC) and DBT (by GC) concentrations (Vivanco, 1992).

97 Table 4-4 Yeast extract broth components (YEB) and (www.atcc.org).

Liquid YEB medium

Tryptone 5 g L-1

yeast extract 1g L-1 Solid YEB medium

nutrient broth, 5g L-1

sucrose 5 g L-1 The same as the liquid with the

MgSO4·7H2O 0.49g L-1 addition of Agar 8 g L-1

All ingredients were dissolved in

water with pH 7.2

Liquid ATCC medium Solid ATCC medium Nutrient Broth (BD cat 234000) 8.0g

L-1 Nutrient Agar (BD 213000) 23g L-1 DI Water 1000 ml DI Water 1000 ml Final pH 6.8

Figure 4-30 a Single-Vial of Agrobacterium rhizogenes from ATCC.

Figure 4-31Agrobacterium rhizogenes growth in 2 different media right YEB left ATCC medium.

Figure 4-32 Contamination in Agrobacterium growth in the revival step before transfer the cells to the suspension culture of the bacteria for inoculation.

YEB ATCC

Figure 4-33 Suspension culture of Agrobacterium growth in 2 different media YEB and ATCC medium to prepare the bacteria inoculation.

A B C

Figure 4-34. Infecting the leaves by A. Making a wound. B. Using fine needle to inject the bacteria suspension culture into the leaves C. Using fine needle to inject the bacteria suspension culture into the roots.

99 Chapter 5 Theoretical Background for the Kinetic Modelling

Research on plant cell suspension cultures goes back a few decades. Although there were extensive investigations on the use of plant cell cultures for the production of primary and secondary metabolites in the 1980s (Pais et al., 1988, M. Pais, 1988), the lack of understanding of the plant molecular biology, metabolism and physiology resulted in low product yields which held back large scale applications. The potential of plant cell cultures for the production of pharmaceutical compounds, flavours, pigments, fragrances and oils under controlled conditions without seasonal, geographical and political influences is still very attractive. Furthermore, their use for the synthetic seed production via somatic embryogenesis is very important for some agricultural, forestry and horticultural applications where seed germination is very difficult. With advances in “omics” science and technology, it is now timely to revisit plant cell biotechnology not only for these applications but also for the sustainable production of platform chemicals, biofuels such as biodiesel, germplasm preservation and artificial seed production of endangered plant species, novel pharmaceuticals and heterologous protein production (Liu, 2015), (Yuan, 2015).

Understanding cell growth and product formation is crucial for developing strategies for large scale applications of plant cell cultures. With renewed interest in the search for novel plant secondary metabolites, natural bioactive compounds, novel pharmaceuticals, preventative healthcare products (such as plant stanols), personal care products (such as plant stem cells in cosmeceuticals) and heterologous proteins from “green factories”, a kinetic model that can predict plant cell culture behavior is a powerful complementary tool to “omics” research and technologies. Some of the early kinetic models for plant cell suspension cultures were reported for apple cells (Pareilleux, 1980) and Catharanthus roseus (van Gulik, 1993), (Bailey, 1989). Several kinetic models have been developed for the production of secondary metabolites by plant cell cultures such as the production of indole alkaloids by C. roseus (Bailey, 1989) phenolic compounds by Nicotina tabacum (Shibasaki, 1995 ) vitamin E by Carthamus tinctorius (Takeda, 1998) and anthocyanin by strawberry cells (Zhang, 1998 ) .A kinetic model was developed to predict nutrient requirement during growth of Eschscholtzia californica cell suspension as well as C. roseus and Daucus carota hairy roots cultures (Cloutier, 2008).

100

Mathematical models of biological systems are very useful tools. Such models can be used: • In interpretation and prediction of experimental results,

• In experimental design and planning,

• For studying and understanding (elucidating) the biological, chemical and physical phenomena and their integration in a process or system,

• For equipment and process design.

• For scale-up/scale-down.

• For control and regulation.

• For product formulation.

• For altering the existing system by setting targets and strategies for genetic, physiological, metabolic and biochemical/bioprocess engineering.

• For scientific and technological knowledge and advancement.

There can be many different types of mathematical models. Kinetic models involve rate laws and mass balances. In this case, the kinetic model involves the biological system of the cell suspension cultures of Nicotiana tabacum. It is an unstructured model that is all the cells are treated as a homogeneous collection with the following assumptions:

• All the living cells have the same size, shape, mass, age, physiology and metabolism. • All the living cells have the same growth environment such as the temperature, nutrient concentrations and pH which assumes a well-mixed vessel. The kinetic model used in this work was developed over the years by the PhD students of F. Mavituna for the plant cell suspension cultures of Taxus spp (Abd-Karim, 2007), Arabidopsis (Mohammed Puad, 2011) and Nicotiana tabacum (Yu, 2016). The relevant literature reviews are presented in these works, and therefore, in this chapter, only the theory relevant to this kinetic model will be summarised. The model expressions were derived based on Michaelis- Menten enzyme kinetics, Monod growth model and the basic kinetic rate laws commonly used in biochemical engineering textbooks (Bailey and Ollis, 1986; Doran, 2000; Mavituna and Sinclair, 2008).

101 5.1 Some Fundamental Concepts

For the kinetic model, it is necessary to define the relevant fundamental concepts such as the volumetric and specific rates, as well as yields before using them in the rate laws and mass balances.

Volumetric rates as defined below depend on the concentration of viable biomass (xv) in the control volume, V of the vessel or bioreactor in which they are cultured.

(퐴푚표푢푛푡⁡표푓⁡cell⁡growth,or⁡푎⁡푐표푚푝표푢푛푑⁡푝푟표푑푢푐푒푑,표푟⁡푐표푛푠푢푚푒푑) 푉표푙푢푚푒푡푟푖푐⁡푟푎푡푒푠 = (Eq. 5-1) (푈푛푖푡⁡푡푖푚푒)(푈푛푖푡⁡푣표푙푢푚푒)

Volumetric rates depend on the “amount of viable cells” in the bioreactor. In order to account for the amount of cells or “normalise” the volumetric rates on a unit biomass concentration basis, specific rates are defined for the growth, product formation and substrate uptake. Specific rates are obtained by dividing the volumetric rate by the viable cell concentration.

푉표푙푢푚푒푡푟푖푐⁡푟푎푡푒 푆푝푒푐푖푓푖푐⁡푟푎푡푒 = (Eq. 5-2) 퐵푖표푚푎푠푠⁡푐표푛푐푒푛푡푟푎푡푖표푛

Volumetric growth rate: rx

푎푚표푢푛푡⁡표푓⁡푏푖표푚푎푠푠⁡푝푟표푑푢푐푒푑 푟 ≡ ⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡(Eq. 5-3) 푥 (푢푛푖푡⁡푣표푙푢푚푒)(푢푛푖푡⁡푡푖푚푒)

Specific growth rate: µ

푟 푎푚표푢푛푡⁡표푓⁡푏푖표푚푎푠푠⁡푝푟표푑푢푐푒푑 휇 ≡ 푥 = (Eq. 5-4) 푥푣 [(푢푛푖푡⁡푣표푙푢푚푒)(푢푛푖푡⁡푡푖푚푒)](푐표푛푐푒푛푡푟푎푡푖표푛⁡표푓⁡푣푖푎푏푙푒⁡푏푖표푚푎푠푠)

Where xv is the viable cell concentration.

Volumetric biodesulfurization (DBT uptake) rate: rDBT

푎푚표푢푛푡⁡표푓⁡퐷퐵푇⁡푐표푛푠푢푚푒푑 푟 ≡ ⁡⁡ (Eq. 5-5) 퐷퐵푇 (푢푛푖푡⁡푣표푙푢푚푒)(푢푛푖푡⁡푡푖푚푒)

Specific biodesulfurization (DBT uptake) rate: qDBT

푟퐷퐵푇 푎푚표푢푛푡⁡표푓⁡퐷퐵푇⁡푐표푛푠푢푚푒푑 푞퐷퐵푇 ≡ = ⁡⁡⁡ (Eq. 5-6) 푥푣 [(푢푛푖푡⁡푣표푙푢푚푒)(푢푛푖푡⁡푡푖푚푒)](푐표푛푐푒푛푡푟푎푡푖표푛⁡표푓⁡푣푖푎푏푙푒⁡푏푖표푚푎푠푠)

102 Volumetric substrate uptake rate: rS

푎푚표푢푛푡⁡표푓⁡푠푢푏푠푡푟푎푡푒⁡푐표푛푠푢푚푒푑 푟 ≡ ⁡⁡⁡⁡⁡ (Eq. 5-7) 푆 (푢푛푖푡⁡푣표푙푢푚푒)(푢푛푖푡⁡푡푖푚푒)

Specific substrate uptake rate: qS

푟푆 푎푚표푢푛푡⁡표푓⁡푠푢푏푠푡푟푎푡푒⁡푐표푛푠푢푚푒푑 푞푆 ≡ = ⁡⁡⁡ (Eq.5-8) 푥푣 [(푢푛푖푡⁡푣표푙푢푚푒)(푢푛푖푡⁡푡푖푚푒)](푐표푛푐푒푛푡푟푎푡푖표푛⁡표푓⁡푣푖푎푏푙푒⁡푏푖표푚푎푠푠)

For a batch culture at constant temperature and volume, a kinetic model can be developed using • Individual mass balance equations for live cells, dead cells (this will be ignored), substrates and products

• Rate equations based on rate laws for growth, death (this will be ignored), consumption of substrates (and DBT)

within the control region (which is the culture vessel, in this case, assuming ideal mixing).

input  output    generation    consumption  accumulation  to    from      within  within  or  control volume control volume +   + control volume  −   − control volume = depletion  by    by        by    by  within  flow or    flow or        reaction    reaction  control volume mass transfer  mass transfer  (Eq. 5-9)

In (Eq. 5-9), the basis for the mass balance may involve simply the amounts such as kg, or rates such as kg h-1 or kg m-3 h-1. Although the SI units are used in this theory chapter, in the application of the model and the presentation of the results g/L and ppm are used following the conventions of the relevant literature.

The hypotheses for our model were based on the observed experimental results depicted in the figures presented in the results and discussion Chapter 7.

103 In the mass balances for the Nicotiana tabacum cell suspension cultures in this work, Michaelis-Menten kinetics for enzymic reactions and Monod kinetics for cell growth are used.

5.2 Michaelis-Menten Kinetics for Enzymatic Reactions

Most media for plant cell cultures contain sucrose as the carbon and energy substrate. The model includes sucrose hydrolysis by the cell-wall invertase enzyme into hexoses (glucose and fructose) Figure 5-1 and consumption of these hexoses at different rates to support cell growth.

Figure 5-1 Hydrolysis of sucrose into glucose and fructose by the invertase enzyme. Michaelis-Menten equation describes the rate of an enzymatic reaction in relation to the rate limiting substrate concentration as:

v S v = max K + S M   (Eq. 5-10)

where: v : the rate of reaction, vmax : the maximum rate of reaction,

[S]: the substrate concentration,

KM : the Michaelis-Menten (or saturation) constant.

The derivation of Michaelis-Menten equation is well documented in the biochemistry and biochemical engineering text books.

104 v S v = max K + S The inverse of M   (Eq. 5-10) will give the Lineweaver-Burk equation as:

1  K  1 1 =  M  + v  v  S v  max  max (Eq. 5-11) or double reciprocal plot as 1/v against 1/S. This plot is used to find the value of vmax and KM.

The y-intercept of this plot gives the value of 1/vmax while the x-intercept gives the value of -

1/KM. Shortcoming of Lineweaver-Burk plot in Figure 5-2is for demonstration in real data, there are longer analytical errors in lower substrate concentration that is in higher 1/S values in the Lineweaver-Burk plot. And example analytical result cluster near the origin of the Lineweaver-Burk plot.

1 Slope = K /V 1/V M max / Velocity 1 max

-1/K M

-1 0 1 2 3 4 1/[Substrate]

Figure 5-2 Lineweaver-Burk plot. 5.3 Monod Kinetics for Growth

Cell metabolism is made up of hundreds of sequential, branched and parallel biological reactions that are normally catalysed by enzymes. The production of these enzymes themselves are an important aspect of metabolism. It can be assumed that growth is the result of hundreds of such enzyme-catalysed reactions. One of these enzyme-catalysed metabolic reactions may become the rate controlling step for growth. This is the basis of the classical Monod equation for cell growth.

105 The relationship between the specific growth rate and a growth rate limiting substrate concentration S proposed by Monod states that:

S  = max (Eq. 5-12) (K S + S)

where

-3 -1 rx is the volumetric rate of cell growth, kg cells m h

-1  max is the maximum specific growth rate, h S is limiting substrate concentration, kg substrate m-3

-3 K S is the saturation constant, kg substrate m

-3 xv is the viable cell concentration, kg cells m

5.4 Maintenance Energy Concept

The total energy required to maintain the concentration gradients which usually exist between the interior and the exterior of cells, and to drive turnover reactions in which labile cell components are continuously re-synthesised is usually referred to as the maintenance energy requirement, being used only to maintain the cell in a viable state and not to produce cell material.

The volumetric rate of consumption of substrate to provide the energy for maintenance is -3 -1 written as rSm (kg substrate m h ), and the generally accepted kinetic expression for the maintenance energy requirement is: r = m x Sm S v (Eq. 5-13)

-1 -1 where, mS is the maintenance (rate) constant (kg substrate kg cells h ).

5.5 Substrate Uptake and Yield Factors (Yields)

Living cells require substrates for three main functions: a. To synthesise new cell material

b. To synthesise extracellular products

106 c. To provide the maintenance energy necessary

i. To drive the synthetic reactions

ii. To maintain concentrations of materials

iii. To drive recycling (turnover) reactions within the cell.

Thus growth, substrate utilisation, maintenance and product formation are all intimately related and the expression for volumetric rate of substrate utilisation, rS, can be written as r = r + r + r S Sx Sp Sm (Eq. 5-14)

Where: rSx is the volumetric rate of substrate consumed for cell growth rSP is the volumetric rate of substrate consumed for the synthesis of extracellular products rSm is the volumetric rate of substrate consumed for cell maintenance

In simple stoichiometrically balanced chemical reactions, the product formed is related to reactant consumed through a stoichiometric yield. When the reactions become complex involving multiple reactants, multiple products and multiple side, parallel or branched reactions, it is difficult to calculate theoretically the stoichiometric yields. Similarly, when the even more complex nature of biological metabolic reactions involving microbial, animal and plant cell cultures are considered, it is very rare that stoichiometric yields can be calculated and used. Instead, experimentally observed yields or yield factors are calculated and used in the models. Such yields or yield factors are defined as follows:

푌′ = ∆푧 = 퐴푚표푢푛푡⁡표푓⁡푧⁡푝푟표푑푢푐푒푑 (Eq.5-15) 푧/푆 ∆푆 퐴푚표푢푛푡⁡표푓⁡푆⁡푐표푛푠푢푚푒푑⁡ Here, z can be the biomass, x, or a product, P, and S can be one of the substrates used for the ′ production of z. Yield factors for biomass on substrate, 푌푥/푆 and for a secreted product on ′ substrate, 푌푃/푆 can be written as

107 ′ ∆푥 푑푥/푑푡 푟푥 푌푥/푆 = = ⁡ = (Eq. 5-16) ∆푆 푑푆/푑푡 푟푆

′ ∆푃 푑푃/푑푡 푟푃 푌푃/푆 = = ⁡ = (Eq. 5-17) ∆푆 푑푆/푑푡 푟푆

Using yield factors, the volumetric rate of a carbon and energy substrate can be written as

r r r = x + P + r S Y ' Y ' Sm x / S P / S (Eq. 5-18)

5.6 A Kinetic Model for Batch Cultures of Nicotiana tabacum Cell Suspensions

The kinetic model was based on the observations of the batch concentration profiles of Nicotiana tabacum cell suspension cultures growing in MS medium containing sucrose which in some experiments also included DBT. Sucrose was hydrolysed in the medium in to glucose and fructose which were simultaneously and concomitantly taken up by the cells but at different rates. In some plant cell cultures, it was reported that glucose inhibited the uptake of fructose but when there was sucrose in the medium this inhibition was not observed. In the experimental results reported in this work, fructose inhibition by glucose was sometimes present (observed as higher fructose concentrations in the medium) and sometimes not. In fact, the inhibition of fructose uptake by glucose was reported for Glycine max (Klerk- Kiebert et al., 1983), D. carota (Krook et al., 2000, Kanabus et al., 1986) and Phaseolus vulgaris (Botha and O'Kennedy, 1998). Therefore, an inhibition term was included in the volumetric uptake rate of fructose and depending on the experimental observations, the inhibition term could be ignored in the kinetic model. Each batch time-course was therefore divided in to three phases:

Batch Phase 1: Sucrose is present in the medium while it is being hydrolysed in to glucose and fructose (all three sugars are therefore present in the medium)

Batch Phase 2: Sucrose is absent in the medium because it is completely hydrolysed but either glucose or fructose or both are present in the medium.

Batch Phase 3: No sugar is left in the medium.

108 The unsteady state mass balances in Batch Phase 1 are represented by Eq.5-19 and Eq.5-22. Mass balance for sucrose gives the rate of sucrose depletion due to hydrolysis as Eq, 5-19: dS S = −r = q x = −V x Eq. 5-19 dt s s v max K + S v S

The mass balance for glucose gives the rate of change of glucose concentration in the medium as a result of formation from sucrose and simultaneous consumption by the cells according to Eq. 5-20:

dG  G  1 = r − r = Y r −  μ x  Eq. 5-20 dt G G G / S S  max K + G v  ' x  G  Y x / G

The mass balance for fructose gives the rate of change of fructose concentration in the medium as a result of formation from sucrose and simultaneous consumption by the cells according to Eq.5-21:

  dF  F  1 = r − r = Y r − μ x Eq.5-21 dt F F F / S S  max K + F v  ' x  F  Y x / F

The mass balance for biomass gives the rate of biomass production from glucose and fructose consumption according to Eq.5-22 dx   v G F = r = μ x = μ  + x Eq.5-22 dt x v max K + G K + F v  G F 

The unsteady state mass balances in phase 2 of the batch culture are represented by Eqs. From Eq.5-23 to Eq 5-26, the mass balance on glucose gives the rate of change of glucose concentration in the medium according to Eq.5-23

109   dG  G  1 = r = − μ x Eq.5-23 dt G  max K + G v  ' x  G Y x / G

The mass balance on fructose gives the rate of change of fructose concentration in the medium according to Eq.5-24       dF  F  1 = −r = − μ x  Eq.5-24 dt F max   v ' x   G  Y x / F  K 1+  + F   F  K     ig  

The mass balance on biomass gave the rate of change of cell concentration as a result of growth with the concomitant consumption of glucose and fructose according to Eq.5-25

      dx  G F  v = r = μ x = μ + x dt x v max  K + G    v Eq.5-25  G  G    K 1 +  + F  F  K    ig  

The DBT concentration in the batch cultures decreased very rapidly. Using the definition of volumetric and specific rates, Eq. 5.5 and 5.6 were used for DBT mass balance to obtain Eq. 5.26 :

푑퐷퐵푇 = −푟 = −푞 푥 Eq.5-26 푑푡 퐷퐵푇 퐷퐵푇 푣

The numerical values of dS/dt, dG/dt, dF/dt, dDBT/dt and dxv/dt at different times during the batch experiments were calculated using the mid-point slope method of the experimental batch culture profiles (Mavituna and Sinclair, 2008). Then, the numerical values of these rates of change of concentrations with time are equal to the volumetric rates according to Eq.5-19 to Eq.5-25, which can then be used to calculate the specific rates. Lineweaver-Burk plot can then be constructed using the specific rate and substrate concentration to obtain the maximum specific rate and the saturation constant. The value of the maximum specific

110 growth rate, µmax can also be estimated from the linear regression of the semi-logarithmic cell dry weight against time plot using the experimental data. The differential equations resulting from the mass balances in the batch culture, Eq.5.19 to 5.26, were written in MATLAB 2017a with built in ODE45 solver based on a sixth stage, fifth order Runge-Kutte method.

5.7 Summary As explained in the introduction to this chapter, mathematical models are useful tools for several reasons. The kinetic model for Nicotiana tabacum cell suspension cultures as described here was applied to the batch cultures to predict the concentrations of cells, sucrose, glucose, fructose and DBT at different batch times. The application of the kinetic model is presented in Chapter 7.

111

Chapter 6 Results and Discussion: Initiation of Various Plant Cell and Hairy Root Cultures

The use of microorganisms for biodesulfurization process to remove sulfur from crude oil without losing the fuel value has been an active research topic since the 1970s (Kodama, 1970). Biodesulfurization was achieved successfully with a range of bacteria and some fungi (Crawford, 1990) at laboratory scale as mentioned in section 2.8. Biodesulfurization PathwaysBiodesulfurization pathways in bacteria have been elucidated and the enzymes identified. After all this research however, for some important reasons that are mentioned in Chapter 1Chapter 8, there is still no large-scale application of microbial biodesulfurization. Sulfur is important for all living cells including plants in several aspects; it is a macronutrient required for cell growth and development (Takahashi, 2001). Several plants are able to utilize sulfur compounds for their metabolic needs. Since some preliminary experiments in Mavituna’s group indicated the biodesulfurization ability of plant cell cultures such as Arabidopsis thaliana and Armoracia rusticana, in this work additional research was performed by molecular docking experiments which confirmed the existence of enzymes in Arabidopsis thaliana, Armoracia rusticana and Nicotiana tabacum (one of the novel aspects of this work), equivalent to the enzymes of 4S pathway in bacteria. Furthermore, using the identified enzymes and various databases, the metabolic pathway reactions with stoichiometric and redox balances for biodesulfurization were collated. This chapter therefore, starts with the results of molecular docking experiments which is then followed by the experimental strategy used for the initiation of plant cell and hairy root cultures.

The experimental strategy for investigating the biodesulfurization potential of plant cell culture was divided into two stages; the first stage involves the initiation of the cultures for all plants: Arabidopsis thaliana, Armoracia rusticana and Nicotiana tabacum as depicted in Figure 6-1. The results of cell and hairy root culture initiation experiments of Stage 1 are explained later in this chapter. The second stage is presented schematically in Figure 7-1 which will be explained in Chapter 7.

112

Plants for Initiation of Cultures

Arabidopsis Nicotiana Armoracia thaliana tabacum rusticana

Seed germination, callus initiation from Callus initiation from seedlings, suspension cultures from callus leaves, suspension cultures

Bulking up of the cell suspension cultures Initiation of hairy roots from leaves and roots Selection of healthy suspension cultures for subsequent experiments

Contamination Nicotiana tabacum suspension of sub-cultured cultures hairy roots

Figure 6-1 Experimental strategy for investigating the biodesulfurization potential of plant cell culture (Stage 1)

113 6.1. Results of Molecular Docking for Biodesulfurization Enzymes

DBT, DBTO, DBTO2, and HBPS ligands were docked to NADP(H) Reductase, Alfalfa feruoyl coenzyme A 3-O-methyltransferase, and AtDHNAT1, a 1,4-dihydroxy-2-naphthoyl- CoA thioesterase as described in Section Chapter 0. The resulting docking predictions were viewed and analysed using the Swissdock server plug in UCSF Chimera Figure 6-2. For comparison, the same procedures were repeated with 4S pathway enzymes in bacterium Rhodococcus erythropolis. These enzymes were DszC, DszA and DszB.

(a1) (a2)

(b1) (b2 )

Figure 6-2 (a) Binding of DBT molecule to the binding site of NADP (H) Reductase, and (b) Binding of HBPS to the binding site of Alfalfa feruoyl coenzyme A 3-O-methyltransferase. Where, a1 and b1 is Cartoon representation, while a2 and b2 is hydrophobic surface representation.

114 The lowest binding energies (Kcal/mol) and ∆G (Kcal/mol) values were recorded. The ∆G (Kcal/mol) data were used to calculate the theoretical affinities values by using the equation ∆G = - RTlnKD, where ∆G = standard change in Gibbs free Energy, R = universal gas constant (8.31 JK-1Mol-1), T = Temperature in Kelvin, and LnKD = Natural log of KD.

Molecular docking results for plant cells and an micoorganizm (Rhodococcus erythropolis) to be compared with plant calls are summarised in Table 6-2Table 6-1 and

Figure 6-3. From the tables the initial dissociation constants (KDs) were calculated by substituting the ∆G (Kcal/mol) values into the Equation ∆G = - RTlnKD. The KDs values were then converted to affinity constant(Ka= 1/KD).

The data shows that plants have the enzymes for biodesulfrization as in 4S pathway in bacteria albeit with different sensitivity and selectivity. An enzyme Alfalfa feruoyl coenzyme A 3-O-methyltransferase exist in Nicotiana tabacum, 1,4-dihydroxy-2- naphthoyl-CoA thioesterase in Arabidopsis thaliana, HRP in Horseradish and NADP(H) Reductase in all plants. NADP (H) Reductase it is the enzyme which is equivalent to NADP (H) and DszC in bacteria. In fact, stating from DBT to HBPS these 4 enzyme can work in each of the 4 steps. Alfalfa feruoyl coenzyme A 3-O-methyltransferase is more specific in Nicotiana tabacum to the last step of the pathway and it is 60 times better in converting HBPS to HBP comparing with its activity in the first three step of the pathway. In addition, Alfalfa feruoyl coenzyme A 3-O-methyltransferase in Nicotiana tabacum is 7.82 times more efficient than DszB enzyme in micoorganizm like Rhodococcus erythropolis towards the oxidation of HBPS to HBP. These results showed that plant cells enzymes are much better in biodesulfurization pathway compared to those in micoorganism. Also, Nicotiana tabacum enzymes can desulfurize DBT more efficient compared to Arabidopsis thaliana and Horseradish. Therefore, experimentally Nicotiana tabacum cells were used in the next chapter to confirm that plant cells can grow in the present (by consuming) sulfur compound like DBT and its intermediate Sulfur compounds. Depend on these results biodesulfrization pathway in plant can be predicted like 4S pathway in microorganisms as seen in Figure 6-4.

115

Table 6-2 Summary of Molecular Docking Results in plant cells Binding energy (Kcal/mol) ∆G (Kcal/mol) Affinity (microMolar)-1 PDB.ID 4hfm 1sus 4k02 HRP 4hfm 1sus 4k02 HRP 4hfm 1sus 4k02 HRP

ligands DBT 4.470 12.041 14.066 11.20 -6.746 -6.130 -5.208 -6.23 0.0858 0.030 0.006 0.04

DBTO 6.856 11.735 16.775 14.24 -6.843 -6.493 -5.987 -6.49 0.100 0.056 0.023 0.06

DBTO2 2.383 6.534 14.170 10.55 -6.784 -6.364 -5.898 -6.25 0.091 0.045 0.020 0.04

HBPS 24.267 11.979 23.481 30.15 -6.889 -8.579 -7.617 -6.47 0.109 1.878 0.371 0.05

PDB.ID=Protein data bank ,4hfm = NADP(H) Reductase, 1sus = Alfalfa feruoyl coenzyme A 3-O-methyltransferase, and 4k02 = AtDHNAT1, a 1,4-dihydroxy-2-naphthoyl-CoA thioesterase, HRP = Horseradish peroxidase

Table 6-1 Summary of Molecular Docking Results in micoorganism (Rhodococcus erythropolis )

Binding energy (Kcal/mol) ∆G (Kcal/mol) Affinity (microMolar)-1 PDB.ID DszC DszA DszB DszC DszA DszB DszC DszA DszB Ligands DBT 13.24 13.02 14.36 -6.06 -6.00 -5.91 0.03 0.02 0.02 DBTO 8.97 13.95 12.82 -6.75 -6.40 12.82 0.09 0.05 0.04 DBTO2 9.45 10.96 16.57 -6.18 -6.33 -5.84 0.03 0.04 0.02 HBPS 19.80 27.93 27.40 -7.52 -6.18 -7.36 0.32 0.03 0.24 PDB.ID=Protein data bank , DszC= Dibenzothiophene monooxygenase, DszA = Dibenzothiophene sulfone monooxygenase, DszB =2'- hydroxybiphenyl-2-sulfinate desulfinase

116

Figure 6-3 Graphical presentation of the theoretical affinity constants of 4hfm = NADP(H) Reductase, 1sus = Alfalfa feruoyl coenzyme A 3-O-methyltransferase, and 4k02 =AtDHNAT1, a 1,4-dihydroxy-2-naphthoyl-CoA thioesterase, HRP = Horseradish peroxidase, DszC= Dibenzothiophene monooxygenase, DszA = Dibenzothiophene sulfone monooxygenase, DszB =2'-hydroxybiphenyl-2-sulfinate desulfinase, toward DBT, DBTO, DBTO2, and HBPS ligands using the data presented in Table 6-2Table 6-1.

117

Figure 6-4 The proposal of 4S for biodesulfurization pathway in plant cells. Where 1sus Nicotiana tabacum enzyme, 4k02 in Arabidopsis thaliana, HRP in Horseradish and 4hfm in all plants and it is the enzyme which is equivalent to NADP (H) and DszC in bacteria, DszB specific for last step to convert HBPS to HBP.

6.2 Metabolic Reactions for Biodesulfurization

After the confirmation by molecular docking that plants have enzymes that can perform the same reactions of microbial 4S biodesulfurization pathway, DBT degradation reactions were searched in KEGG database and the following reactions of Figure 6-5 were collated that include stoichiometric and redox balances (Kegg.com, last access 2018)

118 Biodesulfrization Reactions

1- Dibenzothiophene + Reduced FMN + Oxygen + dszC <=> Dibenzothiophene-5-oxide + FMN

+ H2O

2- Dibenzothiophene-5-oxide + Reduced FMN + Oxygen <=> Dibenzothiophene-5,5-dioxide +

FMN + H2O

3- Dibenzothiophene-5,5-dioxide + 2 Reduced FMN + Oxygen <=> 2'-Hydroxybiphenyl-2-

sulfinate + 2 FMN + H2O

119

4- 2'-Hydroxybiphenyl-2-sulfinate + H2O <=> 2-Hydroxybiphenyl + Sulfite

Figure 6-5 Metabolic reactions for biodesulfurization that show stoichiometric and redox balances. 6.3 Results of Plant Cell Suspension Culture Initiation and Maintenance

Arabidopsis thaliana and Nicotiana tabacum were initiated from the seeds as seen Figure 6-6 Figure 6-7 in these two figures all steps start from seeds for Arabidopsis thaliana; which has smaller seeds than Nicotiana tabacum. After the seeds have germinated, the leaves are green and fresh and the plant already has the features of a whole plant. Horseradish leaves were collected from the pot-grown plant Figure 6-8. These were subsequently transferred to a callus medium; which is a solid medium that contains the materials needed to maintain and transform the nascent plant into friable forms, for a period of three to four weeks before transfer it to liquid suspension medium .

During this period, the size of the plant (now calli) increased dramatically. To ensure that nutrients are easily taken up by calli. The whole Arabidopsis thaliana plant starts to create callies , then they were chopped into small pieces to increase their surface area. This ensured they were well-nourished and they grew steadily. Once they increased in size, the process was repeated and the calli were transferred to a new medium (sub-cultured). As the calli become more friable, it was observed that their colour gradually changes from green to yellow. This happens especially in A. thaliana Figure 6-6 ; which turns yellow earlier than the other plants (Nicotiana tabacum and Horseradish) Figure 6-7Figure 6-8, indicates exhaustion of nutrients. When new nutrients are supplied through sub-culturing, the green colour is restored. However, Nicotiana tabacum were creating the callus faster than Horseradish as shown in Figure 6-10 the Nicotiana tabacum Petri dishes were the one, which have water drops on the top of the dishes this indicates the cells are alive and active. The callus sub-culturing is important for two major reasons: to ensure that the cells are kept viable throughout the length

120 of the experiment and secondly, to use as a stock that serves as backup in the case where there is a contamination during the course of the experiment and there is a need to reuse new cells also where a new experiment is to be initiated. The maintenance of the callus is a very important step because the callus has different stages as seen in Figure 6-11, therefore by monitoring the callus the change of the medium or the cells colour can tell when the cells need subculture or which callus are ready to transfer it to the suspension medium. In order to continue the experiment, suspension culture of the calli needs to be prepared and by so doing we transferred the cells from the solid callus medium in Petri dishes into a liquid medium in flasks , the liquid medium has the same compositions as that of the solid medium but only without nutrient agar. The callus culture and suspension culture was carefully observed frequently to see if there is growth of contaminants as such in certain cases there were unusual growth of contaminants Figure 6-12 that were easily observed in culture and they were quickly discarded and replaced by preparing replacement cultures (Yaqoub, 2012); (Krishnan, 2009). The frequency of contamination increased dramatically after two accidents in the laboratory where the cultures were kept; the ceiling tiles collapsed on to the Petri dishes and shake flasks. Furthermore, the filter of the laminar flow cabinet used for sterile transfers stopped functioning but unfortunately since this was on an annual test contract, the failure of this equipment was not spotted until almost all the cultures were lost because of contamination. The cells increased in sizes and numbers and when the nutrients were exhausted, they were sub-cultured on a new suspension medium. At this point, i.e. when the nutrients have diminished, as in the case of calli, the cells exhibit some physical characteristics such as colour changes. However, once the cells are now in a new medium, they continue to grow, turn green, and condensed water droplets were also observed; which indicates the cells are alive. The cell growth patterns observed under different light intensities were different as better growth was seen when the light was brighter than the growth observed with dim light. (Puad, 2011) also observed lush green in cells grown under cycled light condition in suspension cultures that involved about 12 cycles of sub-culturing (Neumann et al., 2009). If all steps are done in aseptic conditions, from obtained the leaves to reaching the suspension culture, either from seeds or explant then the obtained cells should be maintained carefully to start the biodesulfrization experiments. Occasionally, cell aggregates increased in the size without breaking up, which caused difficulty in the measurement of fresh and dry weight. Therefore, the sieving technique was the best solution to obtain fine cell aggregates for any batch experiment (Figure 6-13Figure 6-14).

121

A.thaliana seeds against a mill-metric ruler. The seeds of A.thaliana in the Petri dish.

The whole plant after taken out from the seed germination medium Healthy growth of A.thaliana leaves and shoots. (leaves, shoots and roots).

Callus was cut into small pieces to A.thaliana Callus is formed around increase the surface area for better nutrient uptake. the explants.

After some time of the sub culturing cells start to turn yellow Figure 6-6 The initiation of Arabidopsis thaliana cell suspension culture starting from the seeds.

122

N.tabacum seeds against a mill-metric ruler. Healthy growth of N.tabacum leaves and shoots

N. tabacum Callus is formed around Initiation of N.tabacum cell suspension culture. the explants.

Figure 6-7 The initiation of Nicotiana tabacum cell suspension culture starting from the seeds.

Figure 6-8 Armoracia rusticana (Horse Radish) growing in compost in a pot.

123

Figure 6-9 The initiation of Horseradish cell suspension culture starting from the leaves collected from the whole plant.

124

Figure 6-10 The plant leaves starting to friable to create the callus, Nicotiana tabacum seems to be more active and faster than Horseradish.

Figure 6-11 In different stages of Nicotiana tabacum callus initiation and maintenance.

125

Figure 6-12 Nicotiana tabacum callus and suspension cultures easily get contaminated

A. contamination in callus B. contamination in suspension culture.

126

Figure 6-13 Nicotiana tabacum plant cells aggregates before sieving.

Figure 6-14 Nicotiana tabacum fine cell aggregates after sieving.

127 6.4 Initiation of Horseradish and Nicotiana tabacum Hairy Roots

The first report that roots may contribute significantly to secondary metabolism in the whole plant was the work of (Dawson, 1942), so hairy roots, which are caused by infection of the soil microorganism Agrobacterium rhizogenes, can be used to produce secondary metabolites. A major disadvantage of extracting phytochemicals from plant cell cultures is the affect that this chemical has on the growth, yield and survival of the plant, which can also be caused by the environmental stress of the extraction procedure itself. An alternative phytochemicals source is HRCs (Hairy root cultures), due to their genetically stable nature, biomass production and comparable biosynthetic capacity to the plant root. The phytochemicals that typically produced in HRCs include alkaloids, phenolics, and terpenoids. HRCs in fact produce a larger amount of phytochemicals in comparison to callus and cell cultures (Vivanco, 1992).

As mentioned, another method to produce plant materials for secondary metabolite production is the culture of shoots, roots, and whole plants. However, these may grow slowly and cause problems in large-scale cultivation, which do not occur in cell cultures. Agrobacterium rhizogenes –transformed hairy roots can synthesize the same components to roots of plants, and have a fast growth rate in hormone-free medium. Hairy root cultures have been studied and efforts made to commercialize the secondary metabolites produced in this method, in a bioreactor (Ono and Tian, 2011, Choi et al., 2008). In literature, the growth condition was changed depending on the species of plants, and different plants varied in their wound response (Saravanakumar et al., 2012). For example, the explant of Withaferin somnifere can be immersed with Agrobacterium rhizogenes for 10 to 20 minutes and then bacteria eliminated with antibiotic of 250mg/l cefotaxime for three days and then placed in the light /dark (16/8h) condition. Also, for Nicotiana tabacum (Kunder and Parasharami, 2014) use 500mg/l cefotaxime (24h) to eliminate the bacteria used for hairy roots induction. (Kastell et al., 2013) and (Shiao and Doran, 2000) infected Arabidopsis thaliana roots by leaving the roots in Agrobacterium liquid culture for one day. To eliminate the bacteria the roots can be left in hormone-free half strength MS medium supplemented with 3% sucrose and 0.2g/l cefotaxime at 26oC in the dark for two to three weeks.

128 (John et al., 2009) infected tea leaves and they use YEB medium for bacteria growth as shown in Table 4-4 . The leaves were left with bacteria for three hours in the dark then in the 250mg/l cefotaxime for 48 hours in the dark.

Other antibiotics have been used to element the bacteria with slightly difference in the conditions. For instances, (Karimi et al., 1999) reported that A.thaliana was infected by Agrubacterium and the antibiotic used was carbenicillin at a concentration of 300mg\l. Moreover, Catharanthus roseus leaves have been infected by Agrobacterium, and were left in the bacteria liquid at 25C° in the light all the time. Then, to eliminate the bacteria, the leaves were transferred to MS free hormone medium supplement with 0.5g/l carbenicillin (Vivanco, 1992). The same antibiotic, carbenicillin, was used with Horseredish leaves which were immersed in bacterial liquid for 10 minutes. The excess bacteria were then removed by using filter paper and placed in solid medium and kept it at 25°C in fluorescent light for three days. After that, the bacteria can be eliminated by placing it in MS free hormone medium with antibiotic of 500휇푔/ml carbenicillin, vancomycin 200휇푔/ml at 25°C in the light. After two weeks, the roots were transferred to MS free hormone liquid medium in the dark. To regenerate the hairy roots, the roots were transferred from the light to the dark. The roots appeared after one week (Noda et al., 1987).

The hairy roots infection can be done not only with the intact plant but it can be done by infecting the callus culture, as mentioned in (Kastell et al., 2013) , through the same steps. The medium was MS free hormone half strength medium which was supplemented with 0.4mg/l 2, 4-D. To get hairy roots from the calli, bacteria can be left on the calli for three days. After eliminating the bacteria, the calli can be kept in the dark at 25°C and sub- culturing every four weeks in the same conditions (Zhu et al., 2014).

In this work, to obtain the optimal conditions for hairy roots initiation different methods of infection and conditions were attempted ( Table 6-3Table 6-4). First of all, the bacteria culture should be ready as mentioned in Figure 4-33 and then the sterilized plant is infected with Agrobacterium rhizogenes by using a needle or by making small wounds and injecting the bacteria sample into the plant Figure 4-34. As shown in Table 6-3 the hairy roots infection process, which was done by making small wounds failed many times, when the leaves were left in the cefotaxime antibiotic for 24 hrs. The leaves

129 withered and when the time was reduced for an hour the leaves got heavy contamination as seen in Table 6-3(Exp 1Exp 2). The infection was done using fine needle to inject the bacteria suspension culture and the leaves were left for 24 hours. Also, they have withered (see Exp 3). (Habibi et al., 2016) suggested a method to culture the plant tissue in the dark after the hairy root disease infecting bacteria have been eliminated but this did not work in this experiment, as can be seen in (Exp 4), as keeping the leaves in the dark made them lose their green colour. In addition, (Vivanco, 1992) mentioned that instead of using antibiotic to eliminate the bacteria, the plants can be placed under high temperature at 38oC. As shown in Table 6-4 (Exp 6), the three plants Arabidopsis thaliana, Armoracia rusticana and Nicotiana tabacum could not survive the high temperature when sterilized with this method. The roots themselves can be infected besides the plant leaves, so the roots were infected for more growth. Furthermore, it was suggested that the roots be weighed before culturing to be able to record the growth of the hairy roots (Hedviga Komarovskáa, 2009a). But this attempt was not successful as seen in Exp 7. The root explants were kept in the dark and it was difficult to eliminate the bacteria from the root.

The time for eliminating the bacteria with antibiotic should neither be too short nor too long, as mentioned in (Hedviga Komarovskáa, 2009b). After infection, the plant was placed in an antibiotic solution for one day. However, in this work, elimination of bacteria was tried for 20 minutes and there was contamination but not as heavy as the wounding infection see Exp 5.

The successful infection that gives good hairy roots was with plants that were left for 1 hour in 250mg/l cefotaxime; which is the suitable concentration for eliminating bacteria. After eliminating the bacteria, the leaves were placed in the incubation condition (16/8 h light /dark). Room temperature of 25 to 28°C was the optimal condition. In addition, 250mg/l cefotaxime was the suitable concentration to eliminate the bacteria Exp 8.

After two to three weeks, the roots appeared and grew fast. When they became longer, the roots were separated from the leaves (Figure 6-21). It is then transferred to the hormone-free MS liquid medium and left to grow (Figure 6-22). The roots became contaminated and it was decided to stop the hairy root work. However, from this study, at least a suitable technique was developed for the successful production of the hairy roots from Armoracia rusticana and Nicotiana.tabacum.

130

Table 6-3The table is shown different methods of infection and conditions have been attempted to obtain the optimal conditions for hairy roots initiation

Step1 Roots initiation in Step2 Step3 Experiment No Condition Infection after surface Nicotiana tabacum Eliminate the bacteria Washing sterilization and Horseradish Infect the leaves by making wound and (16/8 h light /dark) and leave the leaves in Exp 1 immerse the plant in The leaves vitrified and room temperature of 250mg/l cefotaxime for died bacteria suspension 25-28oC 24 h. culture for 1 day Figure 4-34 (16/8 h light /dark) and leave the leaves in Wash it from the Exp 2 Heavy Contamination room temperature of - 250mg/l cefotaxime for antibiotic by using Figure 6-15 25-28oC 1 h. Distilled water and Infect the leaves by use place it in hormone free

(16/8 h light /dark) and fine needle to inject the leave the leaves in MS solid medium. Exp 3 room temperature of bacteria suspension 250mg/l cefotaxime for The leaves vitrified and 25-28oC culture 24 h. died Figure 4-34 leave the leaves in The leaves lost their Exp 4 In the dark and room 250mg/l cefotaxime for green color and nearly temperature of 25-28oC 20min. died Figure 6-16.

131

Table 6-4 Continue the table is shown different methods of infection and conditions have been attempted to obtain the optimal conditions for hairy roots initiation

Step1 Roots initiation in Step2 Step3 Experiment No Condition Infection after surface Nicotiana tabacum Eliminate the bacteria Washing sterilization and Horseradish Non-heavy (16/8 h light /dark) and leave the leaves in Exp 5 Contamination Figure room temperature of 25- - 250mg/l cefotaxime for 6-17 28oC 20min.

leave the leaves in Exp 6 (16/8 h light /dark) and No hairy roots - 250mg/l cefotaxime for 38oC Figure 4-18 20min. Wash it from the Infect the Roots of antibiotic by using Horseradish by use fine Distilled water and leave the roots in Exp 7 In the dark and room needle to inject the place it in fee hormone Contamination and 250mg/l cefotaxime for wilted the roots. temperature of 25-28oC bacteria suspension MS solid medium. 20min. Figure 6-19 culture

leave the leaves in (16/8 h light /dark) and Positive results (the root Exp 8 250mg/l cefotaxime for room temperature of 25- - start to grow 1 h. 28oC after 10days)Figure 6-20.

132

Figure 6-15Heavy Contamination for (Exp2).

Figure 6-16 The leaves lost their green color and nearly died (Exp4).

Figure 6-17Non-heavy Contamination (Exp5).

133

Figure 6-18No hairy roots (Exp6).

Figure 6-19 Contamination and wilted the roots. (Exp7).

Figure 6-20 Positive results (the root start to grow after 10 days) right Horseradish, lift Nicotiana tabacum (Exp8).

134

Figure 6-21 The growth was successful of the roots in Nicotiana tabacum leaves which appear after a week and then continue grow , ready to separate from the leaves.

Figure 6-22 The separated hairy roots from the leaves in the solid MS free hormone medium to liquid MS free hormone medium.

135 6.5 Summary

In summary, the cell culture initiation was performed for three types of plants Arabidopsis thaliana, Armoracia rusticana and Nicotiana tabacum. Nicotiana tabacum was chosen as the model of plant to continue with for biodesulfrization experiments due its faster growth and the callus and suspension culture look healthier than other plants. Moreover, it was confirmed by the results of molecular docking that Nicotiana tabacum cells have the more efficient ability to desulfurize sulfur compounds (DBT) than microorganisms (e.g Rhodococcus erythropolis) by following 4S pathway as shown in Figure 6-3 , which has the feature of keeping the carbon skeleton of sulfur compounds as well as the value of fuel/oil and calories still intact and commercially useful for the petroleum industry. The enzyme in Nicotiana tabacum the Alfalfa feruoyl coenzyme A 3-O-methyltransferase is 7.82 times more efficient than DszB enzyme in micoorganizm like Rhodococcus erythropolis towards the oxidation of HBPS to HBP. This indicates that the use of plant cell cultures will be the novel development in biodesulfrization field. Finally, the hairy roots were the great idea to help the biodesulfurization process by using them as self-immobilization systems in column bioreactors. Unfortunately, after initiating the hairy roots from Nicotiana tabacum and horseradish successfully, heavy contamination caused the loss of all hairy root suspension cultures. A great deal of time was spent in developing the technique and the conditions for the successful initiation and initial growth of hairy roots but the hairy roots experiment was not repeated due to the lack of time in the project.

136

Chapter 7 Results and Discussion: Use of (Nicotiana tabacum) Cell Suspension Cultures for Biodesulfurization

In Results and Discussion: Initiation of Various Plant Cell and Hairy Root Cultures, the initiation and bulking up of Nicotiana tabacum plant cells cultures were reported following the Stage 1 strategy as shown in Figure 6-1. Since only Nicotiana tabacum cell suspension cultures grew well and without contamination, biodesulfurization experiments of Stage 2 reported in this chapter were performed using this plant species. The experimental strategy of stage 2 is shown in Figure 7-1. As shown in this figure, the experiments were performed in MS, MS supplemented with DBT and SFM media supplemented with DBT in the absence or in presence of crude oil in continuous light condition at 25oC, in either shake flasks with cotton or rubber bung (airtight) or in Duran bottles (for safety reasons when using crude oil) placed on orbital shaker at 110rpm. All media had 30g /l sucrose as a sole carbon source which was normally converted to glucose and fructose by cells’ enzymes in the medium. Glucose and fructose were consumed by cells simultaneously. In SFM it was necessary to add DBT as the sole sulfur source. The cultures were inoculated at 10% v/v. Most of the batch experiments were run for 3 to 4 weeks, and when there were enough plant material 2 flasks were sacrificed for taking a sample for the growth measurement every 3-4 days to increase the reliability of the results obtained. All biomass concentrations are reported as (g dry weight)-1 and sucrose, glucose and fructose in g/l and DBT and 2-HBP in ppm. These different experimental conditions with their codes are summarised in Table 7-1 with their associated subsection and figure numbers. What is not shown in table 7-1 are; application of the kinetic model introduced in Chapter 5 to the experimental results after each major subsection of the chapter.

137

Nicotiana tabacum suspension cultures

Normal MS growth medium Sulfur-free MS medium

Addition of different amounts of DBT

Control batch Addition of Effect on cell experiments: Crude Oil viability Growth, sugar uptake

Subsequent cell growth Batch biodesulfurization after experiments: contact Growth, sugar uptake, Effect of no air with crude DBT consumption exchange on growth oil for (Duran bottles) various lengths of time

Figure 7-1 Experimental strategy for investigating the biodesulfurization potential of Nicotiana tobacum cell culture (Stage 2)

138

Table 7-1 The table shows different experimental conditions with their codes summarised with their associated subsection and figure numbers. Section Medium Figure No. Flask Duran DBT Oil Exp. Code No. 7.1.1 MS Figure 7-2 ✓ MS

MS Figure 7-3 ✓ MS.D

SF Figure 7-4 ✓ 100 SF.100

SF Figure 7-5 ✓ 100 SF.100.D

7.1.2 MS Figure 7-7 ✓ 100 MS.100

MS Figure 7-7 ✓ 200 MS.200 7.1.3 MS Figure 7-10 ✓ 200 MS.200/6h

SF Figure 7-11 ✓ 100 SF.100/6h

7.2.1 FDA test for the effect of crude oil on cell viability 7.2.2 MS Figure 7-25 ✓ MS 7.2.3 MS Figure 7-27 ✓ 100 ✓ MS.100.D.O

SF Figure 7-28 ✓ 100 ✓ SF.100.D.O

SF Figure 7-30 ✓ 200 ✓ SF.200.D.O

SF Figure 7-31 ✓ 400 ✓ SF.300.D.O

SF Figure 7-32 ✓ 300 ✓ SF.400.D.O

139 7.1 Nicotiana tabacum Growth and Biodesulfurization in Aqueous Media

7.1.1 Effect of Air Exchange on Nicotiana tabacum Cell Culture in MS and SFM with 100ppm DBT The normal growth conditions reported in the literature for Nicotiana tabacum cell suspension cultures involve one of the commonly used basal media, such as MS in this research, in culture vessels that allow air exchange ie, oxygen and carbon dioxide, such as shake flasks with sterile cotton bungs loosely covered with aluminium foil, at 25oC with continuous mixing. The experiment performed under these conditions therefore, and reported in Figure 7-2 (A and B) serve as the control in this research. Furthermore, in this section, the effect of air exchange was examined in MS medium and SFM with 100ppm DBT because later when the crude oil will be added, the experiments cannot be run in normal flasks which will allow air exchange including the vapours from crude oil due to safety regulations. Therefore, Duran bottles or any container with air-tight closure (no air exchange) should be used.

In MS normal medium in shake flasks with air exchange, three experiments are reported, Figure 7-2(A) and (B) which show similar growth trends. The first sample taken 72 h after inoculation indicated growth reaching the maximum biomass concentration of 19.3 g/l for (A) and 15.2 g/l for (B) at 432 h. However, for same medium with air tight flasks, as can be seen in Figure 7-3 the growth was slower with the maximum biomass concentration of 14.4 g/l at 450 h. This was despite the fact that the air-tight culture had a slightly higher inoculum concentration. This indicates the slightly adverse effect of not having enough oxygen in the headspace.

Sucrose concentration decreased while glucose and fructose increased initially indicating sucrose hydrolysis but since the concentration of glucose and fructose were not the same and at stoichiometric yields of sucrose hydrolysis, this indicated that cells were consuming glucose and fructose concomitantly with sucrose hydrolysis. This will be reflected in the two phases of the batch used in the application of the kinetic model reported at the end of this section (Chapter 07.1.4 Nicotiana tabacum Kinetic Model Applications for Cultures in Aqueous Media. There was almost no sucrose left in the medium by 216 h and glucose and fructose were consumed by 288 h as shown in Figure 7-2 (A) and (B). Despite the absence of any sugars, cells seem to continue their growth until 432h. It is a well-known fact that plant

140 cells can take up sugars as well as other nutrients such as nitrate, phosphate and store them sugar in their vacuoles for future use.

In the batch culture in Duran bottle Figure 7-3 again sucrose hydrolysis to glucose and fructose was observed by decrease in sucrose concentration while glucose and fructose concentrations increased in the medium (phase 1 in the kinetic model applied later). Sucrose was exhausted by 144 h soon after which glucose and fructose concentrations started to decrease with some fluctuation indicating uptake as shown by accompanying growth until about 432 h. Cell concentration decreased after this time and since there was a slight increase in both glucose and fructose concentrations in the medium, it may be speculated that some cells autolysed releasing these in to the medium.

141 35 (A)

5 Biomass and Sugar Conc. (g/l) Sugar andBiomass

0 0 100 200 300 400 500 Time (h) Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l

35 (B) 30

Biomass and Conc. and (g/l) Sugar Biomass 5

0 0 50 100 150 200 250 300 350 400 450 500 Time (h)

Biomass DW (g/l) Sucrose (g/l) Glucose (g/l) Frucrose (g/l)

Figure 7-2 Nicotiana tabacum batch culture control experiment in MS medium in flasks with cotton bung allowing air exchange (Code.MS). A- The experiment run with one sample each time, B- The average results of two other experiments with same conditions as A.

142 40

Biomass and Sugar Con. (g/l) Sugar andBiomass 5

0 0 100 200 300 400 500 Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l

Figure 7-3 Nicotiana tabacum batch culture in MS medium in Duran bottles with no air exchange (Code.MS.D). This experiment was run twice Figure 7-4 (A) and (B) show the time course of batch culture in shake flasks in SFM supplemented with 100ppm DBT in shake flasks. The culture in (A) was inoculated with cells that were adapted to growth in sulfur-free MS (SFM) supplemented with 100ppm DBT as the main sulfur source for several subcultures. The culture depicted in Figure 7-4 (B) on the other hand was inoculated with cells straight from normal MS medium. Despite these differences in inocula, both cultures indicated almost no growth. The adapted inoculum increased from 5g/l to 7.8g/l and non-adapted inoculum had no obvious growth. MS normal medium contain sulfate salts as sulfur sources equivalent to sulfur in 300ppm DBT. This may mean that these cultures were both deficient in sulfur. Sulfur is the fourth important macronutrient in plant cell cultures after nitrogen, phosphorus and potassium. Without enough sulfur, nitrogen assimilation and metabolism as well as photosynthesis also suffer and therefore plant cells do not reach their full growth potential. Sulfur deficiency symptom in whole plants appear as yellowing of the new and then the old growth (Promix.com, 2018, Bond, 2017), It can therefore be assumed that 100ppm DBT was not enough for healthy cell growth in the cultures. Later, when crude oil was included in the SFM with 100ppm DBT, cells grew well (Figure 7-28) due to the possible presence of other sulfur compounds in the oil.

143 Sucrose concentration decreased slowly as shown in Figure 7-4 (A) until 360 hours when it became zero. There was 11g/l glucose and fructose in the medium at the start which may have come from sucrose degradation during autoclaving as sucrose concentration at the start was 23g/l. However, if the stoichiometric equimolar conversion of sucrose hydrolysis is considered, 1g sucrose would yield 0.53g of glucose and fructose each. Therefore, from 7g/l sucrose degradation, 3.71g/l of glucose and fructose should be produced. The discrepancy (instead of 11g/l) may be due to the presence of dead cells in the inoculum autolysing and releasing their glucose and fructose contents in to the medium. Cell wall bound sucrose hydrolysis enzymes would still be active even if the cells were dead. Sucrose would therefore continue to be converted to glucose and fructose which are taken up by the living cells in the inoculum which then grow to reach 7.8g/l. Similar arguments can be made for the concentrations of sucrose, glucose and fructose in Figure 7-4(B) but since there was no growth these sugar concentrations did not drop to zero even after 216 h. The sugars that appear to be consumed must be taken up by the living cells either for storage in the cells’ vacuoles which cell do especially during nutritional stress such as nitrogen, phosphate and sulfur deficiency or consumed for the energy needs associated with biodesulfurization or other maintenance energy needs. The same medium SFM with 100ppm DBT was also used in air tight flasks. In this condition, the growth started early reaching a maximum biomass concentration of 12.7g/l at 72 hours and then the growth started to decrease when sucrose was completely consumed. Although there was still glucose and fructose in the medium at that time, most probably in the air-tight conditions in Duran bottle, the culture ran out of oxygen which was made worse (compared to normal MS in Duran bottle, Figure 7-3) because of sulfur deficiency as seen in Figure 7-5.

DBT concentration decreased from 100ppm to 8.48ppm in the culture with SFM adapted inoculum (A) and to 12ppm in the non-adapted inoculum case (B) in 72h. In Figure 7-6 it can be seen that the DBT degradation indeed occurred by detecting the presence of 2-HBP production during the growth of (Nicotiana tabacum) using Gibss assay. According to the 4S pathway of biodesulfurization, every 1 mol of DBT gives 1 mol of 2-HBP, (Yaqoub, 2012). Since the concentrations of DBT and 2-HBP are reported in ppm (following the convention in oil refinery related desulfurisation reports), it is necessary to convert ppm in to Molar (M) or milli Molar (mM) units. Assuming that the density of the medium is equal to that of water (1 g/ml),

144 100ppm DBT = 54mM DBT = 54 mM 2-HBP = 93ppm 2-HBP

Figure 7-6 indicates that for the culture in flasks with air exchange using sulfur-free MS supplemented with 100ppm DBT (SF.100), 2-HBP production fluctuated; but it was slightly higher than 85.1ppm 2-HBP which corresponds to the degradation of 100 – 8.48 = 91.5ppm DBT. The highest concentration of 2-HBP was 97ppm. Also presented in Figure 7-6 is the 2- HBP concentrations in the culture in Duran bottles with no air exchange in sulfur free medium supplemented with 100ppm DBT (SF.100.D) using inoculum adapted to growth in SF.100. Since 100ppm DBT almost disappeared from the medium, the maximum concentration of 2-HBP expected was 93ppm. However, the maximum 2-HBP concentration was 74ppm, lower than the expected 93ppm. These small discrepancies must be because of the analytical errors in the sample analysis. Alternatively, the inocula for these two experiments were adapted to growing in sulfur-free medium with 100ppm DBT supplementation and hence the cells might have some 2-HBP stored in their vacuoles which was then released in to the medium increasing the 2-HBP concentration in the flasks with air exchange. Oxygen is needed in the metabolic reactions of 4S pathway reactions (section 6.2 Metabolic Reactions for Biodesulfurization) and the culture in Duran bottles, perhaps did not have enough oxygen to complete these pathway reactions even if the DBT was converted halfway through the 4S pathway. This may explain the lower than expected values for the concentration of 2-HBP. The kinetic model is applied to these culture results in Section 7.1.4 Nicotiana tabacum Kinetic Model Applications for Cultures in Aqueous Media

145 35 (A) 120

30 100 25 80 20 60 15 40

10 DBT Conc. (ppm) Conc. DBT 5 20

0 0 Biomass and Sugar Conc. Conc. (g/l) Sugar andBiomass 0 100 200 300 400 500 Time (h) Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

(B) 35 110

30 90 25 70 20 50 15 30 10 (ppm) Conc. DBT 5 10

Biomass and Sugar Con. (g/l) Sugar andBiomass 0 -10

0 50 100 150 200 250

Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-4 Nicotiana tabacum batch culture in SFM medium with 100ppm DBT in flasks with cotton bung allowing air exchange (Code.SF.100). A- The experiment run with one sample each time, B- Another experiment with same conditions as A.

146 35 120

30 100

25 80 20 60 15 40 10 (ppm) Conc. DBT

5 20 Biomass and Sugar Conc. Conc. (g/l) Sugar andBiomass

0 0 0 100 200Time (hrs)300 400 500

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-5 Nicotiana tabacum batch culture in SFM medium with 100 ppm DBT in Duran bottles with no air exchange. This experiment was run in duplicate (Code.SF.100.D).

120

100

HBP Conc. (ppm) HBP Conc.

- 2

0 0 100 200 300 400 500 600 Time (h) SFM.D.100ppmDBT SFM.100ppmDBT

Figure 7-6 The production of 2-HBP during the culture of Nicotiana tabacum in SFM.D.100ppm DBT and SFM.100ppmDBT(A).

147

7.1.2. Effect of DBT Concentration on Nicotiana tabacum Growth in MS Aqueous Medium.

In this section, the effect of DBT with two different concentrations of 100ppm and 200ppm was examined in the MS medium in shake flasks with normal air exchange. There were not enough cells obtained in this stage to run both of experiments in duplicate. In both cases, there was an obvious long lag phase with 100ppm Figure 7-7 (A) and 200ppm DBT Figure 7-8 (A); and growth started from 144 hours and reached the maximum biomass concentrations of 24 and 11.36g/l at 360 and 288 hours, respectively. When these maxima were reached, sucrose was completely consumed in both cultures. In the culture with 100ppm DBT, glucose and fructose were also exhausted but in the culture with 200ppm DBT, about 8 g/l each of glucose and fructose remained in the medium, corresponding to the lower biomass concentration achieved in this culture.

These experiments were repeated in order to check the reproducibility of the results. These are presented as the averages of two experiments in Figure 7-7 (B) and Figure 7-8 (B). Since plant cells behave differently each time depending on the inoculum physiology (age and viability) and concentration, these experiments gave somewhat different results. Culture growth trend was similar for the cases of MS.100 but in the case of set (B) the biomass concentration reached only a maximum of 13g/l. Therefore, although all the sucrose was consumed, some glucose and fructose remained in the cultures (Figure 7-7B). DBT was taken up faster with 100ppm DBT compared with 200ppm. As Figure 7-7 (A) shows for 100 and Figure 7-8 (A) for 200ppm DBT, the DBT concentrations were 4.94ppm in 360 hours and 0.75ppm in 288 hours, respectively at the same time of reaching the maximum biomass concentration. And to confirm that DBT is consumed by the plant cells, 2-HBP production was detected as seen Figure 7-9. In both cases, the DBT concentration remaining in the medium was very low. However, theoretically, 93ppm and 186ppm 2-HBP should be expected from the conversion of 100 and 200ppm DBT, respectively. The maximum concentrations of 2-HBP were 74 and 120ppm for 100 and 200ppm DBT conversion, respectively. Plant cells seems to store some of 2-HBP when it is converted from DBT and then release it again to the medium.

148

Kinetic model was applied on the best grow example, which was in MS and MS.100ppm DBT in air exchange flask. However, SFM.100ppm DBT was an example of unsuccessful growth. Table 7-2 shows the stoichiometric and kinetic parameters relating to this growth and sucrose consumption in both experiments for MS normal medium experiment obtained maximum volumetric rate of sucrose hydrolysis (g/lh) and Michaelis constant of sucrose hydrolysis (g/l) from Figure 7-14,7-15 and 7-16. Moreover, the same parameters were obtained for MS.100ppm DBT from Figure 7-17,7-18and 7-19 for SFM.100ppmDBT Figure 7-20 Figure 7-21. The determination of the values of these parameters is explained in the kinetic model development Chapter 5.

149 35 (A) 120

30 100

25 80 20 60 15 40

10 (ppm) Conc. DBT

5 20 Biomass and Sugar Conc. Conc. (g/l) Sugar andBiomass

0 0 0 100 200 300 400 500 Time (h) Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT (ppm)

25 120 (B) 100 20

80 15 60 10 40

(ppm) Conc. DBT 5 20

Biomass and Sugar Conc. (g/l)Conc. Sugar and Biomass 0 0 0 100 200 300 400 500 600 700 Time (h)

Biomass DW (g/l) Sucrouse (g/l) Glucose (g/l)

Fructose (g/l) DBT Con. (ppm)

Figure 7-7 Nicotiana tabacum batch culture in MS medium supplemented with 100ppm DBT in flasks with cotton bung allowing air exchange (Code.MS.100). A- The experiment run with one sample each time, B- The average results of two other experiments with same conditions as A.

150 35 (A) 250

30 200 25

150 20

15 100

10 (ppm) Conc. DBT 50

Biomass and Sugar Conc. (g/l)Conc. Sugar and Biomass 5

0 0 0 100 200 300 400 500 Time (h) Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

30 250 (B)

25 200

20 150 15

100 10 Conc.(ppm) DBT 50 5 Conc. (g/l) Sugar andBiomass

0 0

0 100 200 300 400 500 Time (h)

Biomass DW Con (g/l) Sucrose (g/l) Glucose (g/l)

Fructose (g/l) DBT Con. (ppm)

Figure 7-8 Nicotiana tabacum batch culture in MS medium supplemented with 200ppm DBT in flasks with cotton bung allowing air exchange (Code.SF.200). A- The experiment run with one sample each time, B- The average results of two other experiments with same conditions as A.

151 140

120

100

HBP Conc. (ppm) HBP Conc. -

2 40

0 0 100 200 300 400 500 Time (h) MS + 100 ppm DBT MS+200ppm DBT

Figure 7-9 The production of 2-HBP during the culture of Nicotiana tabacum in MS.100ppm DBT (A) and MS.200ppmDBT(A).

7.1.3 Six-hourly Sampling of Nicotiana tabacum Cultures in MS Aqueous Medium Supplemented with 200ppm DBT and in SFM Aqueous Medium Supplemented with 100ppm DBT

In the Sections 7.1.1 Effect of Air Exchange on Nicotiana tabacum Cell Culture in MS and SFM with 100ppm DBT7.1.2. Effect of DBT Concentration on Nicotiana tabacum Growth in MS Aqueous Medium.it was observed that although the DBT was taken up very quickly by the plant cells, in some experiments this was not accompanied by the simultaneous good growth such as SFM.100 and MS.200 in Figure 7-4 and 7-7 respectively. There were also some fluctuations observed in the concentrations (cell, sugars, DBT and 2-HBP). In those experiments, samples were taken approximately every 72 h over 500 h of the batch course. To investigate what was happening, it was decided to take samples at shorter intervals, every 6 h instead of every 72 h for 92 h duration for a few experiments. 92 h was chosen because it was the period during which most of sucrose was converted and most sugars consumed and most of the DBT disappeared but without much change in the cell concentration. The chosen media were SFM.100 and MS.200 in shake flasks. In both experiments the cells seem to experience a lag phase during the 92 hours as shown in Figure 7-10Figure 7-11. Following the initial rapid conversion of sucrose in the first 6 h, in the subsequent periods, sucrose,

152 glucose and fructose concentrations fluctuated in both experiments (Figure 7-10 Figure 7-11). Such fluctuations were also observed by other researchers in plant cell cultures (Mavituna, personal communication); (Yu H 2016) However, DBT decreased rapidly from 200 to 19ppm and from 100 to 2.4ppm in 6 hours and remained mostly around these values without obvious fluctuation. 2-HBP appearance in the medium on the other hand, showed a noticeable level of fluctuation as can be seen in Figure 7-12. However, the concentration of 2-HBP was higher in MS medium with 200ppm DBT as expected because MS medium already has 300ppm sulfur in addition of 200ppm DBT unlike SFM with 100ppm DBT as the only sulfur source. In SFM.100 in Figure 7-11 2-HBP fluctuations seem to follow the fluctuations in sucrose concentration.

In this set of experiments, the relationship between the fresh weight and the dry weight was established in Figure 7-13. As can be seen, for the same dry weight concentration, the MS.200 culture has cells with higher fresh weight than those in SFM.100. A higher fresh weight implies larger cells with larger vacuoles containing more aqueous liquid (water) for a given cell dry weight. Having a limited sulfur source as discussed before, may have reduced the cell growth reaching its full potential. Nevertheless, such relationships help determine the dry weight or the fresh weight once one of these is known.

153 35 250

30 200 25

20 150

15 100 10 50 (ppm) Conc. DBT

5 Biomass and Sugar Conc. Conc. g/l Sugar andBiomass 0 0 0 20 40 60 80 100 Time (h) Boimass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-10 Nicotiana tabacum batch culture (dry weight) g L-1, sugar and DBT analysis. The samples were collecting every 6 hours for 90 hours’ batch, in MS medium supplemented with 200ppm DBT (Code.MS.200/6h). 35 150

30 130 110 25 90 20 70 15 50

10 (ppm)Coc. DBT 30 5 Biomss and Sugar Conc. g/l Conc. Sugar Biomss and 10

0 -10 0 20 40 60 80 100 Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-11 Nicotiana tabacum batch culture (dry weight) g L-1, sugar and DBT analysis. The samples were collecting every 6 hours for 90 hours’ batch, SFM medium supplemented with 100ppm in air exchange flasks (Code.SF.100/6h).

154 180

160

140

120

100

HBP(ppm) Conc.

- 2 40

0 0 10 20 30 40 50 60 70 80 90 100 Time (h)

SFM.100ppmDBT MS.200ppm DBT

Figure 7-12 The production of 2-HBP during the culture of Nicotiana tabacum in SFM.100ppm DBT and MS.200ppm DBT every 6 hours (This experiment was not run in duplicate)

100 90 80 y = 13.155x

60 50 SFM.100 40 y = 10.751x MS200 30 Fresh weight g/l weight Fresh 20 10 0 0 1 2 3 4 5 6 7 Dry weight g/l

Figure 7-13 The relationship between fresh and dry weight (g L-1) in MS 200ppm DBT and SFM 100ppm DBT in every 6 hrs collection time.

155 7.1.4 Nicotiana tabacum Kinetic Model Applications for Cultures in Aqueous Media The numerical values of the kinetic model presented in(Eq.5-19 to 5-26) were obtained by constructing the Lineweaver-Burk plots (Figure7-14,717 and 7-20) for sucrose conversion in the medium, lnx (the natural logarithm of biomass concentration) vs time for µmax for phase 1 and 2 (Figure7-15,7-16,7-18,7-19 and 7-21). The values of the rest of the parameters were either calculated from experimental results or obtained from a previous research student (Yu H 2016) who also worked with Nicotiana tabacum cell cultures. These values are listed in Table 7-2. MATLAB program version 2017a with built in ODE45 solver based on a sixth stage(Appandex1), used to solve the differential equations fifth order Runge-Kutte method resulting from the mass balances in batch cultures with the appropriate initial conditions (ie., concentrations at zero time) and some biological constraints such as non-zero concentrations, no growth when sugar concentration is zero etc. However, not all biological activity terms were used in this model such as the maintenance energy and death kinetics because of the lack of reliable experimental data. The model solution did not predict the experimental results well mainly because of the errors involved in the determination of the values of the kinetic parameters from the experimental results from the plots as mentioned above. Furthermore, the model was relatively basic and more sophisticated models may be needed to express the complex biological activities of the plant cell cultures. The problems associated with heterogeneous nature of the plant cell cultures and their samples render the reproducibility of the experiments difficult. Furthermore, a set of experiments are needed for initial rate experiments with different amounts of initial glucose and fructose concentrations with each experiment run separately and also with different combinations of these sugars in order to determine the sugar uptake kinetics properly. Since plant cells have large vacuoles in which they can store medium nutrients such as sugars as well as nitrogen and phosphorous sources, internal concentrations of such compounds should also be analysed. In this model the values of some kinetic parameters and stoichiometric parameters such as yield of biomass formation from fructose, Monod constant for fructose uptake were obtained from Professor Mavituna’s group who had run separate experiments for sucrose, glucose and fructose. In most cases, the parameter values were adjusted in order to improve the fit of the model and these are indicated in brackets in Table 7-2. The predictions of the kinetic model are compared with the experimental results in Figure 7-22, 7-23 and 7-24 for MS, MS100 and SF.100 , which were chosen as comparable conditions. Although the predictions of biomass and sucrose

156 concentrations are reasonable, the model predicts higher concentrations for glucose and fructose. This may be improved by decreasing the values of yields of biomass on glucose and fructose. Also, the model needs a maintenance energy term which would have helped reduce sugar concentrations.

Table 7-2 Stoichiometric and kinetic parameters relating to (Nicotiana tabacum) growth and Sucrose and DBT consumptions in MS normal medium and SFM medium

Parameters MS MS.100 SFM.100 Sources

X0 (g/l) 2.543 5.454 5.9 19.336 24.46 7.8 X (g /l) In 18 days In 15 days max In12 days 288hr 432 hrs 360 hrs -1 -1 MS (Figure 7-2) P max (g cells l h ) 0.038 0.053 0.0066 SFM.100 (

P (g cells l-1h-1) avg 0.03 0.01 0.0032 Figure 7-4) ∆S (%) 99.32% 100% 99.83% MS.100(Figure 7-7) Y’ (g cells /g X/S 0.6 0.63 0.07 sucrose) qDBT N/A 0.0003 0.00021 MS (Figure 7-15) -1 SFM.100(Figure 7-18) µmax (h ) 0.006 0.001 0.0009 MS.100(Figure 7-21)

MS (Figure 7-16) µ ’(h-1) 0.003 0.0008 0.0003 max MS.100(Figure 7-19) V (g sucrose/(g MS (Figure 7-14) max 0.003 0.008 0.02 cells)(h) SFM.100 (Figure 7-17) Ks (2.5) (0.05) (0.18) MS.100(Figure 7-20) 3.104 K g 7.97 k f 3.104 K g 7.97 K f Y’X/g ((g cells) (g (Yu H 2016) glucose)-1) 0.3 Y’X/f ((g cells) (g fructose)-1) YG/S ((g glucose) (g sucrose)-1) 0.53 YF/S ((g fructose) (g sucrose)-1) -1 kig (g glucose (L) ) (1000000)

157 6000

5000

4000 y = 835.53x + 200.27

3000 1/qs

2000

1000

0 0 1 2 3 4 5 6 1/s

Figure 7-14 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain 푉max⁡and 퐾푀 in MS normal medium in air exchange condition.

2.5

1.5

y = 0.0063x + 0.9408 lnx 1

0.5

0 0 50 100 150 200 250 Time(h)

Figure 7-15 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax'1 in MS normal medium in air exchange condition.

158 3

2.9

2.8 y = 0.0029x + 1.6322

2.7 lnx

2.6

2.5

2.4 0 100 200 300 400 500 Time (h)

Figure 7-16 Semi-log plot of phase 2 for determination of the maximum specific growth rate μmax'2 in MS normal medium in air exchange condition.

700

600

500 y = 6.3423x + 97.487 400

1/qs 300

200

100

0 0 10 20 30 40 50 60 70 80 90 1/s

Figure 7-17 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain 푉max⁡and 퐾푀 in MS normal medium with 100ppm DBT in shake flasks in air exchange.

159 2.5

1.5 y = 0.0023x + 1.4119 lnx 1

0.5

0 0 50 100 150 200 250 Time(h)

Figure 7-18 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax’1 in MS normal medium in air exchange condition supplemented with 100ppm DBT.

3.5

2.5

2 lnx y = 0.0038x + 1.0573 1.5

0.5

0 0 100 200 300 400 500 Time(h)

Figure 7-19 Semi-log plot of phase 2 for determination of the maximum specific growth rate μmax'2 in MS normal medium in air exchange condition supplemented with 100ppm DBT.

160 200 180 160 140 120

100 1/qs 80 y = 9.0079x + 58.095 60 40 20 0 0 2 4 6 8 10 12 14 1/S

Figure 7-20 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain 푉max⁡and 퐾푀in SFM medium in air exchange condition supplemented with 100ppm DBT.

2.5

1.5 lnx 1 y = 0.0024x + 1.3611

0.5

0 0 50 100 150 200 250 300 350 Time (h)

Figure 7-21 Semi-log plot of one phase for determination of the maximum specific growth rate μmax' in SFM medium in air exchange condition supplemented with 100ppm DBT.

161

Figure 7-22 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in MS normal medium with air exchange condition. (Code.MS)

Figure 7-23 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in MS normal medium supplemented with 100ppm of DBT with air exchange condition. (Code.MS.100)

162

Figure 7-24 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in SFM medium supplemented with 100ppm of DBT with air exchange condition.(Code.SF.100)

163 7.2 Nicotiana tabacum Growth and Biodesulfurization in Aqueous Medium with Addition of Crude Oil In advance of running biodesulfrization experiments in the presence of crude oil, two methods of checking viability after contacting with crude oil as explained in section 4.6. Cell Viability Tests Staring from developing washing technique and the first method is FDA staining and the second involves monitoring culture growth subsequent to contacting with crude oil.

7.2.1 FDA Test for the Effect of Crude Oil on Cell Viability As explained in section 4.6.2 Cell Viability Test by Using Fluorescein Diacetate, when a few drops of FDA solution are added to cell samples on microscopy slide, the esterases in the live cells can convert FDA to dissociated fluoresces which fluoresces green under UV at with 490nm wavelength. The dead cells do not have this enzymatic activity and hence do not show fluorescence. Table 7-3Table 7-4 show the photographs of FDA test results obtained from cells contacting with crude oil mixed with MS medium for (0, 30 mints, 1h, 2, 24h,4 day,8 day and 12 day) and cells contacting with crude oil without MS medium for (0 ,1hr and 12day), respectively. In these tables, FDA column show the fluorescing cells and the overlay indicate both fluorescing and non-fluorescing cells. Because of the strict safety requirements involving the use of crude oil, these experiments were performed in the shortest possible time and hence no cell counts in microscopic fields were attempted. Only the fluorescence was taken as an indication to cell viability. Indeed, the cells show reasonably good viability after contacting with crude oil both in the presence and absence of MS medium up to 12 days. This viability is further proven by subsequent culture growth experiments as explained next.

164

Table 7-3 Effect of crude oil on cell viability when its contact with mixture of MS medium in presence of crude oil for various lengths of time.

Time contacting with crude oil and medium With FDA Overlay

0 hour.

30mins.

1 hour

2 hour

1 day

4 day

8 day

12 day

165

Table 7-4 Effect of crude oil on cell viability when its contact with crude oil without medium for various lengths of time. (0h,1h, 12 days).

Time contacting with crude oil only With FDA Overlay

0 hour.

1 hour.

12 days

166 7.2.2 Subsequent Cell Growth in MS Medium for 2 weeks After Contact with Crude Oil for Various Lengths of Time Examine and monitoring Nicotiana tabacum cell growth culture in MS medium subsequent to contacting with crude oil in different length of time (2,4,6,8,24) h. In this experiment, the analysis involved only filtration; to determine the cell dry weight and use HPLC to calculate sugar uptake. For all contacting length of time, Figure 7-25 shows that the longer the cells were contacted with crude oil before being washed, the less growth was experienced. Another observation was that the cells did not reach as great weight as in the previous section experiments in aqueous medium with and without DBT, in with and without air exchange. Must be noted that the plant cells were contacting only crude oil without medium, which can be the most affected factor. The maximum biomass concentration was seen with 2h contact time at 6.1g/l, for the 4h has the same pattern between 336 and 408h a slightly increase was from 3.5 to 4.3g/l. The two graphs showed an almost exponential growth starting with no evidence of the cells entering the death phase in the 408h. That was evident that if the cells were leaving longer and continue to grow and the entering of death phase can be determined. For 6,8 and 24h contacting time, no significant growth was exhibited and but the cells seem to enter the death phase specially with 6 and 8hrs. However, with 24h the cells biomass concentration was fluctuating between 1 and 2.5g/l at 240 to 336h no growth and no sugar up taking. The sucrose concentration in all the contact times except 24h was converted completely to glucose and fructose after 168h. Moreover, the 24h contacting time the sucrose concentration was zero in 72h and converted to glucose and fructose with a higher concentration because the cells were not growing to consume the sugar in the medium.

167

2 hrs contacting time with crude oil 35 7 30 6 25 5

20 4 15 3 10 2

Sugar Conc. Conc. g/l Sugar 5 1 Biomass Conc. Conc. (g/l)Biomass 0 0 0 50 100 150 200 250 300 350 400 450 Time (h)

Sucrose g/l Glucose g/l Fructose g/l Biomass Conc. g/l

4 hrs contacting time with crude oil

35 5 30 4 25 20 3

15 2 10

Sugar Conc. Conc. g/l Sugar 1 5 Biomass Conc. (g/l)Conc. Biomass 0 0 0 50 100 150 200 250 300 350 400 450

Time (h) Sucrose Conc. g/l Glucose Conc. g/l Fructose Conc. g/l Biomass Conc. g/l

6 hrs contacting time with crude oil

35 4 30 3 25 20 2 15 10 1

Sugar Conc. Conc. g/l Sugar 5 0 0 Conc. (g/l)Biomass 0 50 100 150 200 250 300 350 400 450 Time (h) Sucrose Conc. g/l Glucose Conc. g/l Fructose Conc. g/l Biomass Conc. g/l

168 8 hrs contacting time with crude oil 35 4 30 25 3 20 2 15

10 1

Sugar Conc. g/lConc. Sugar 5 Biomass Conc. Conc. (g/l)Biomass 0 0 0 50 100 150 200 250 300 350 400 450 Time (h) Sucrose Conc. g/l Glucose Conc. g/l Fructose Conc. g/l Biomass Conc. g/l

24 hrs contacting time with crude oil 35 3

30 2.5 25 2 20 1.5 15 1 10

Sugar Conc. Conc. g/l Sugar

5 0.5 Biomass Conc. Conc. (g/l)Biomass 0 0 0 50 100 150 200 250 300 350 400 450 Time (h) Sucrose Conc. g/l Glucose Conc. g/l Fructose Conc. g/l Biomass Conc. g/l

Figure 7-25 Nicotiana tabacum culture and sugar up take ,the cells was growing MS normal medium after contacting crude oil in different length of time every two hours (2,4,6,8, 24) h contacting time after wash them by using saline solution.

169 On another hands, Another fact was noticed that the dry cells after contacting crude oil look wilted on the filter paper as seen in Figure 7-26 the dry cells from control experiment crude oil free MS medium compared to cells were growing in MS normal medium after 2hrs contacting time with crude oil. The wilted dry cells were observed in all the later samples regardless of contact time.

Figure 7-26The normal dry cells in crude oil free MS normal medium in the left and in the right the last sample at 408 hrs shows wilted dry cells in MS normal medium after washing from 2hrs contacting time with crude oil.

This could be happened due to the use of saline solution to wash the cells and remove the crude oil. When the cells were washing with saline solution the water inside the cell may move out of the cells to attain equilibrium because saline solution is a hypertonic solution (Study.com.).Therefore, The residual saline solution, which remain in the MS normal medium after washing the cells from the crude oil because there was no method to dry the cells and over time more water was exited the cells that why the later samples seem more wilted. To sum up, the growth can be effected by the length of contacting time the longer the cells were contacted with crude oil, the less the cells grew and the residual saline solution.

7.2.3 Biodesulfurisation Using MS and Sulfur-free MS Media and Crude Oil

In this section, several experiments are reported which involve the addition of 2ml crude oil to 20ml aqueous medium with DBT addition of 100, 200, 300 and 400ppm. Because of the safety concerning crude oil, all these were performed in airtight 100ml Duran bottles or 50 ml shake flasks stoppered with rubber bungs. As a control, MS medium with the addition of 100ppm DBT was used; whereas the rest of the cultures were in sulfur-free MS (SFM) with DBT addition. Except for the control, all inocula came from cultures adapted to growth in sulfur-free medium with 100ppm DBT supplementation.

170 For the MS normal medium as seen in Figure 7-27 there was only some growth; the inoculum concentration was 9.07g/l and the maximum dry weight was 16.4g/l in 144h after which the average biomass concentration decreased and remained around 10g/l. However, in SFM medium towards the end of the batch Figure 7-28 maximum dry weight value of 26.69g/l was obtained at 460h and afterwards, the growth started to decrease. In MS normal medium, Figure 7-27 all sugar concentrations decreased to very low values from 144 hours onward, but since there was no further growth after 144h the cells may take them up but store them in vesicles and use them for the maintenance energy and energy needed for DBT degradation and not for growth. However, in the SFM medium, sucrose was not converted as early as that observed in MS, nevertheless its concentration became zero by 261h. The concentrations of glucose and fructose were zero at the beginning of the batch, then they increased concomitantly reaching their maximum concentrations when sucrose was depleted Figure 7-28 .From their maxima, glucose and fructose concentrations decreased following the increase in biomass concentration.

The standard MS normal medium (Sigma-Aldrich.) contains sulfur in the form of sulfate salts as equivalent to 300ppm sulfur. Crude oil used contained approximately 3140ppm DBT. When 100ppm DBT and crude oil naturally containing some DBT (about 3140ppm) were added to MS, the sulfur concentration increased above 400ppm. The cells consumed the amount of sulfur (from DBT and sulfate) needed for the sulfur metabolism and the rest either was converted stored in the vacuoles or remained in the medium which was detected in GC- FID chromatogram as shown in Figure 7-27. However, with SFM medium, Figure 7-28, which had the DBT and crude oil as the only sources of sulfur, the cells tended to use up most of the DBT. In addition, as evidence of DBT conversion, 2-HBP was found in the medium as the end product of 4S pathway Figure 7-29. The graph shows that in MS medium 2-HBP concentration was more than SFM. This section indicates that SFM.100.D.O medium is better for the cell growth than MS normal medium. Therefore, in next section different concentrations were examine in SFM medium in presence of crude oil.

171 40 120

35 100 30 80 25

20 60

15 40 10 (ppm) Conc. DBT 20

Biomass and Sugar Conc. Conc. g/l Sugar andBiomass 5

0 0 0 100 200 300 400 500 600 Time (h) Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-27 Nicotiana tabacum batch culture in MS normal medium (dry weight) g L-1 and sugar analysis, the growth was in Duran bottle (No air exchange) presence of crude oil and 100ppm DBT (Code.MS.100.D.O).

35 100 90 30 80 25 70 20 60 50 15 40 10 30 20 (ppm) Conc. DBT

Biomass and Sugar g/l Sugar andBiomass 5 10 0 0 0 100 200 300 400 500 600 Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-28Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis, the growth was in Duran bottle (No air exchange) presence of crude oil and 100ppm DBT (Code.SF.100.D.O).

172 300

250

200

150

100

HBP conc. (ppm)HBP conc. -

2 50

0 0 100 200 300 400 500 600 Time (h)

2-HBP in MS.O.D.100 (ppm) 2-HBP in SFM.O.D.100(ppm)

Figure 7-29 The production of 2-HBP during the culture of Nicotiana tabacum in MS and SFM medium with 100ppm DBT in presence of crude oil.

Since the biodesulfurization process in SFM medium supplemented with 100ppm DBT in the presence of crude oil seems to be successful in the previous section; the effect of higher concentrations of DBT, 200 ,300,400ppm, were examined to check how much DBT the plant cells culture could tolerate for biodesulfrization process to occur. The cells grew at the beginning of the batches in both SF.200.D.O and SF.400.D.O and reached the maximum biomass concentration of 11.6g/l at 72h and 18g/l at 144h, respectively. Sucrose was converted to glucose and fructose from the 72 and 144h of the incubation with 200 and 400ppm DBT respectively, as seen in Figure 7-30

35 450 30 400 350 25 300 20 250 15 200 150 10

100 (ppm) Conc. DBT 5 50

Biomss and Sugar Conc. g/l Conc. Sugar Biomss and 0 0 0 100 200 300 400 500 600 Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

173 Figure 7-32. However, culture in SF.300.D.O indicated almost no growth. Sucrose appeared in the medium until very late at 460h and glucose and fructose were detected in the medium from 72h and reached zero in the last sample at 500h Figure 7-31. Moreover, DBT and any sulfur compounds in crude oil can supply the cells with a higher amount of sulfur than the amount added (Esmail Alkhalili, 2017), which may be challenging to calculate. Even if the concentration of DBT decreased and the cells were not growing, the vacuoles of the cells have the ability to store the sulfur compounds which can enter the metabolic pathway at any time (Leustek et al., 2000). The concentrations of DBT in all three batches fluctuated at higher concentrations than those in MS.100.D.O and SF.100.D.O. In addition, 2-HBP concentration in the medium fluctuated as seen in Figure 7-33 with the concentrations of 2-HBP with 200ppm and 300ppm DBT being higher than those in 400ppm DBT. However, with 400ppm DBT the cells may lose their ability to degrade DBT, the cells may store sulfur and keep it in the vacuoles which then leads to less concentration of 2-HBP in the medium.

Error! Reference source not found. lists a comparison of overall DBT biodesulfurization rates and biomass p roductivities for these different experiments in SFM medium with varying amounts of added DBT and 2ml crude oil

DBT remaining Overall BDS Rate Maximum Biomass DBT Conc. (ppm) Productivity (ppm DB converted) in the medium (ppm) (g cells l-1h-1) (g cells l-1h-1) 100 0.006 0.043 0.031 200 1.8 0.133 0.036 300 73 0.166 - 400 0.8 0.18 0.039

174

35 200 30

25 150 20

15 100

10 (ppm) Conc. DBT 50 5

Biomass and Sugar Conc. Conc. (g/l) Sugar andBiomass 0 0 0 100 200 300 400 500 600 Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-30 Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis and 200ppm DBT, the growth was in Duran bottle (No air exchange) presence of crude oil (Code.SF.200.D.O).

175 35

10 (ppm) Conc. DBT

5 Biomass and Sugar Conc. Conc. (g/l) Sugar andBiomass 0 0 100 200 300 400 500 600 Time (h) Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-31Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis, the growth was in Duran bottle (No air exchange) presence of crude oil with 300ppm DBT (Code.SF.300.D.O).

35 450 30 400 350 25 300 20 250 15 200 150 10

100 (ppm) Conc. DBT 5 50

Biomss and Sugar Conc. g/lConc.Biomss andSugar 0 0 0 100 200 300 400 500 600 Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-32Nicotiana tabacum batch culture in SFM medium (dry weight) g L-1 and sugar analysis and 400ppm DBT, the growth was in Duran bottle (No air exchange) presence of crude oil (Code.SF.400.D.O).

176 600

500

400

300

200

HBP Conc. (ppm) HBPConc.

- 2 100

0 0 100 200 300 400 500 600 Time (h) 2-HBP in SF.O.D.100(ppm) 2-HBP in SF.O.D.200 (ppm) 2-HBP in SF.O.D300(ppm) 2-HBP in SF.O.D.400(ppm)

Figure 7-33 The production of 2-HBP during the culture of Nicotiana tabacum in SFM supplemented with 100, 200, 300 and 400 ppm DBT in the presence of crude oil.

177 7.2.4 Effect of Sulfur Compounds Naturally Occurring in the Crude Oil on Cultures in Sulfur-Free-Medium

Sample from the stock crude oil was analysed in GC-FID using column HP-1 as explained Section 4.5 Analytical Techniques which showed that it contained both DBT and 2-HBP. The latter is the final product in the 4S biodesulfurization pathway of sulfur compounds like DBT in crude oil. However, 2-HBP may occur in the crude oil through gradual spontaneous air oxidation of the crude oil a process which is similar to the industrial BDS of crude oil. Crude oil was added to the aqueous sulfur-free medium supplemented with DBT, and during the experiments when samples were taken, the crude oil layer was allowed to coalesce and collect as the top layer which was then separated by carefully pipetting out and then centrifuging to get rid of any aqueous part. These were also analysed for their DBT and 2- HBP contents in the GC-FID with column DB-5MS. There was either no or very low concentrations of DBT in the crude oil samples from the experiment reported in Section 7.2 Nicotiana tabacum Growth and Biodesulfurization in Aqueous Medium with Addition of Crude Oil. The concentrations of DBT in the aqueous medium (Figure 7-28Figure 7-30Figure 7-31

35 450 30 400 350 25 300 20 250 15 200 150 10

100 (ppm) Conc. DBT 5 50

Biomss and Sugar Conc. g/l Conc. Sugar Biomss and 0 0 0 100 200 300 400 500 600 Time (h)

Biomass DW g/l Sucrose g/l Glucose g/l Fructose g/l DBT Conc. (ppm)

Figure 7-32. ) and for 2-HBP in the aqueous medium (Figure 7-33) and crude oil phase (Figure 7-34) can be used to make some approximate mass balances.

178 90 80 70 60 50 40

HBP Conc. HBP(ppm) - 2 20 10 0 0 100 200 300 400 500 600 Time (h)

SF.O.D.100 SF.O.D.200 SF.O.D.300 SF.O.D.400

Figure 7-34 2-HBP concentration in crude oil samples from cultures of Nicotiana tabacum in SFM medium in air tight condition with different concentrations of DBT addition.

Using the GC-FID chromatographs of Section 4.5 Analytical Techniques The stock crude oil had 3140ppm DBT and 207.3ppm 2-HBP. The density of the crude oil was measured as 0.66g/ml. Using the definition of ppm, these mean that crude oil had: 3140 mg DBT/1 000 000 mg crude oil or 3140 mg DBT/1515.15 ml crude oil.

In 2ml crude oil (added to the aqueous medium) therefore, there was 4.15mg DBT. It was assumed that the density of the aqueous medium was approximately 1g/ml. If all this 4.15mg DBT from 2ml crude oil transferred in to 20ml (or 20g) aqueous medium, this meant an additional concentration of 207.3ppm DBT in the aqueous medium. Similar calculations indicated that from 2ml crude oil, the additional 2-HBP in the aqueous medium would be about 25ppm. Since the analysis of the crude oil samples taken from the experiments in Section 7.2.3 Biodesulfurisation Using MS and Sulfur-free MS Media and Crude Oil had no or very low DBT concentrations, it can be concluded that the cultures not only converted the DBT added to the cultures but also almost all of this 207ppm DBT from the crude oil. This can be proved by a simple mass balance on DBT as follows which was performed for SF.100.D.O for demonstration purpose Figure 7-28.

179

mAi = mg DBT in 20 ml aqueous medium at the start of the batch mAo = mg DBT in 20 ml aqueous medium at the end of the batch mOi = mg DBT in 2 ml crude oil at the start of the batch mOo = mg DBT in 2 ml crude oil at the end of the batch md = mg DBT degraded in the batch

Mass balance on DBT: mAi + mOi - mAo - mOo = md

0.002 mg +4.145 mg -0.001 – 0 = 4.147 mg DBT degraded =0.0225 mmol DBT degraded

This means that the amount of DBT added to the aqueous medium was very small compared to the amount of DBT naturally occurring from the crude oil and the culture degraded almost all the DBT both from the aqueous medium and the crude oil. A similar mass balance can be made for the 2-HBP as follows: hAi = mg 2-HBP in 20 ml aqueous medium at the start of the batch hAo = mg 2-HBP in 20 ml aqueous medium at the end of the batch hOi = mg 2-HBP in 2 ml crude oil at the start of the batch hOo = mg 2-HBP in 2 ml crude oil at the end of the batch hp = mg 2-HBP produced in the batch

hAi + hOi - hAo - hOo = hP 0 + 0.5 – 0.008 – 0.08 = 0.412 mg 2-HBP produced = 0.00242 mmol 2-HBP produced Since 1 mol DBT degradation gives 1 mol 2-HBP, the values above indicate that cells either kept most of the 2-HBP intracellularly, most probably in the vacuoles or they converted it to other compounds. This is probable because plant metabolism is much more complex than that of the microorganisms and the biodesulfurization pathways in plants have not been studied.

180 7.2.7 (Nicotiana tabacum) Kinetic Model Application for the Biodesulfurization Experiment in Presence of Crude Oil

For the application of the kinetic model, the best growth experiment which was SF.100.D.O. was chosen. The parameters were either estimated from various plots Figure 7-35Figure 7-36Figure 7-37 or taken from other sources as listed Table7-5. The model does not take in to consideration the presence of the crude oil except for indirect effects on the maximum specific rates of Phase 1 and Phase 2. It is obvious from Figure 7-38 that the model does not provide a good prediction of the experimental data. Some recommendation for its improvement are given in Conclusion and Recommendations.

Table7-5 Stoichiometric and kinetic parameters relating to Nicotiana tabacum growth and Sucrose and DBT consumptions in SFM medium supplemented with 100ppmDBT in presence of crude oil

BDS Parameters Sources SFM.100.D.O

X0 (g/l) 13.2 26.7 X max (g /l) In 18day 432hr -1 -1 P max (g cells l h ) 0.031 Figure 7-28 -1 -1 P avg (g cells l h ) 0.02 ∆S (%) 71% Y’X/S (g cells /g sucrose) 0.45 qDBT 9 -1 µmax (h ) 0.003 Figure 7-36 -1 µmax’(h ) 0.005 Figure 7-37 Vmax (g sucrose/(g cells)(h) 0.012 Figure 7-35 Ks (10.33) 3.104 Kg 7.97 Kf -1 Y’X/g ((g cells) (g glucose) ) 0.3 -1 Y’X/f ((g cells) (g fructose) ) (Yu H 2016) - YG/S ((g glucose) (g sucrose) 1) - 0.53 YF/S ((g fructose) (g sucrose) 1) -1 kig (g glucose (L) ) (1000000)

181

350 300 250 200 y = 852.7x + 82.477 1/qs 150 100 50 0 0 0.05 0.1 0.15 0.2 0.25 0.3 1/S

Figure 7-35 Lineweaver_Burk plot of (Nicotiana tabacum) Michaelis–Menten to obtain 푉max⁡and 퐾푀 in SFM medium in no air exchange condition supplemented with 100ppmDBT in presence of crude oil.

3.5

2.5

2 y = 0.0027x + 2.6064

lnx 1.5

0.5

0 0 50 100 150 200 250 Time (h)

Figure 7-36 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax'1 in SFM medium in air exchange condition supplemented with 100ppm DBT in presence of crude oil.

182 3.5

2.5

2 y = 0.0046x + 1.0046 lnx 1.5

0.5

0 0 100 200 300 400 500 600 Time (h)

Figure 7-37 Semi-log plot of phase 1 for determination of the maximum specific growth rate μmax'2 in SFM medium in air exchange condition supplemented with 100ppm DBT in presence of crude oil.

Figure 7-38 Kinetic model solved with MATLAB and experiment data of Nicotiana tabacum in SFM medium supplemented with 100ppm of DBT with air tight condition in presence of crude oil (Code.SF.100.D.O).

183 Chapter 8 Conclusion and Recommendations

The aim of this project was to investigate the biodesulfurization capability of plant cell suspension and hairy root cultures to improve biocatalysts for biodesulfurization of crude oil. Since the use of plant cell cultures was the novel aspect of this research in biodesulfurization field, each phase of this research needed the development of new techniques before the experiments on the plant cell's ability in biodesulfurization. Methods and techniques were developed for callus and cell suspension cultures initiation from Arabidopsis thaliana, Armoracia rusticana and Nicotiana tabacum. Hairy roots were also initiated from Armoracia rusticana and Nicotiana tabacum using Agrobacterium rhizogenes. Cell sieving technique to reduce plant cell aggregate size, since cells were contacted with crude oil, special techniques such as cell washing technique, adding crude oil to the medium under safety requirements were also developed in this research. High Performance Liquid Chromatography (HPLC) was used for the determination of sucrose, glucose and fructose concentrations, Gas Chromatography (GC) was used to monitor the degradation of DBT and 2-HBP production as end-product in biodesulfurization pathway and Spectrophotometer UV by using Gibb's assay to detect 2-HBP and other phenol derivatives and UV microscope to test the cell viability by using fluorescein diacetate. In addition, the Swissdock server and other servers were used for the molecular docking to check the existence of the enzymes for the equivalent 4S biodesulfrization pathway in plant cells.

Since biodesulfrization process was successful with various of microorganisms and 4S biodesulfrization pathway was the best pathway due to removing the sulfur from polycyclic aromatic sulfur compounds; without loss of coal fuel value such as in Rhodococcus erythropois sp. Therefore, by molecular docking (Swissdock server) the equivalent enzymes in this pathway were found in all plants which were tried Arabidopsis thaliana, Armoracia rusticana (horseradish), and Nicotiana tabacum (Nicotiana tabacum). Moreover, this study demonstrated that plant enzymes are more effective comparing with the bacteria enzymes. The most efficient and popular micoorganizm (Rhodococcus erythropolis) in biodesulfurization was compared with (Nicotiana tabacum) in the last step’s enzyme where the 2-HBP (4s pathway dead end-product) and the realised sulfate were appear 1sus enzyme in (Nicotiana tabacum) plant cells can work 7.82 times efficient the than DszB enzyme in (Rhodococcus erythropolis). In the results the affinities for both were 1.878 and 0.24 (μM)-1 respectively.

184

Indeed, molecular docking indicated that plant cells, Arabidopsis thaliana, Armoracia rustican and Nicotiana tabacum had the equivalent enzymes to perform biodesulfurization. However, since only Nicotiana tabacum cell suspension cultures were the healthy ones with good growth characteristics and minimal contamination problems, these were chosen for the biodesulfurization experiments in this work. All plants were growing well in the same conditions and Murashige and Skoog medium until suspension cultures were obtained. However, Nicotiana tabacum was the most healthier indeed it was the faster in growth. Moreover, the original aim included the investigation of biodesulfrization process in plant and as hairy roots part of the plant it can be used to help biodesulfrization process to be more efficient by using it as self-immobilization. This idea was novel as therefore, in the same time of plant initiation and maintenance the hairy roots were initiation when the healthy leaves were obtained after many attempt on Armoracia rusticana (horseradish), and Nicotiana tabacum (tobacco) the successful initiation experiment was when the leaves were infected by use fine needle to inject the Agrobacterium rhizogenes bacteria suspension culture and leave it for 24hrs and then eliminate bacteria by left the infected leaves for 1 hour in 250mg/l cefotaxime and wash the leaves from the antibiotic (cefotaxime) by using distilled water and place it in MS-free hormone nutrient medium in the incubation condition (16/8h light /dark), room temperature of 25 to 28oC was the optimal condition. After the hairy roots were separated from the leaves and placed in MS-free hormone nutrient liquid medium unfortunately the hairy roots were contaminated, which caused the loss of all the roots. Regardless of that, the optimum techniques and conditions to obtain hairy roots were established in this study, which can be followed in the future work.

The experiments in this work were performed either in aqueous Murashige- Skoog (MS) or sulfur-free Murashige-Skoog (SFM) medium. Other components were added, 30g/l sucrose and some of the medium components for plant cultivation such as kinetin, naphthalene acetic acid (NAA), and 2, 4- dichlorophenoxyacetic acid (2, 4-D), which are plant growth regulators are needed for the cells to grow in vitro. in the presence or absence of dibenzothiophene(DBT), as the conventional model sulfur compound, in shake flasks (50ml) or with the same media with the addittion of crude oil in air tight condition e.g. Duran bottles (100ml ) or shake flasks (50ml) stoppered with a rubber bung.

185 Nicotiana tabacum (tobacco) control experiments were in aqueous medium in shake flasks against which the other experimental results could be compared. Airtight vessels had to be used for safety reasons when the experiments involved crude oil. The experimental work can be concluded in two parts. The first part involved the investigation of the growth, sugar uptake and DBT consumption in aqueous medium. The MS.100 experiment had the highest maximum biomass concentration at 24.46g/l in day 15 (360hrs) with maximum biomass productivity at 0.053 (g cells l-1h-1) and at the same time (360 hrs) the sucrose concentration was 0.01g/l while glucose and fructose were higher than sucrose at 0.5 and 0.7g/l respectively, and 95% of DBT was consumed by day 15. However, the concentration of 2-HBP, which was detected by Spectrophotometer UV by using Gibb's assay was 66.3ppm. That shows most of the DBT was converted to 2-HBP. The second part involved the addition of crude oil. Before running the experiments with the addition of crude oil, cell viability test after contacting the cells for different lengths of time with crude oil was done. For this, fluorescein diacetate staining (FDA) under UV microscope was used which showed that cells could stay alive up after 12 days of contacting with crude oil. Furthermore, the cells’ growth after 24h contact with crude oil was tested by washing them and growing them in MS normal medium for around 2 weeks. The results show the cells had reasonable growth from 2hrs contacting up to 24hrs but the less contacting time the better growth. Therefore, the crude oil can be added to the medium with DBT, to investigate how much sulfur the cells can tolerate to grow when crude oil was added. The results show that the lowest amount of DBT, 100ppm, was the best concentration for the cells to grow in. The best growth and DBT degradation was with SFM.100.D.O experiments as seen in Figure 7-28. The Maximum biomass concentration at 26.7g/l in day 21 (432hrs) with Maximum Productivity at 0.031(g cells l-1h-1) and at the same time (432 hrs) all sugars concentrations were completely consumed, 97.7% of DBT was consumed at day 21. However, the concentration of 2-HBP was 46.31ppm. Comparing MS.100 with SFM.100.D.O, the MS normal medium already had 300ppm sulfur from sulfate salts in addition to 100ppm of sulfur from DBT. However with SFM.100..D.O the sulfur was supplemented from100ppm DBT and crude oil, which was equivalent to approximately 200ppm DBT as calculated .

Kinetic model was used to obtained some quantitative comparison between some experiments in different conditions, based on Michaelis-Menten enzyme kinetics for sucrose hydrolysis to glucose and fructose, and Monod growth model on glucose and fructose. The values of some kinetic parameters and stoichiometric parameters were obtained from the experimental data

186 and some from other sources. The model was solved in Matlab and computed concentration were compared with the experimental results. The model predictions were not very good. This was mainly because of the difficulty in obtaining reproducible results with plant cell cultures. Homogeneous inoculation and sampling are difficult because of the aggregated nature of the cultures. Furthermore, it is well known that plant cells can store nutrients and products as well as metabolic intermediates in their vacuoles and release them during the batch. The concentrations in the medium can also show cyclic fluctuations. To make things even more complex, although these cultures are supplied with sugars and hence expected to grow heterotrophically, they can grow photosynthetically when the culture conditions allow. One additional factor that made experiments difficult in this research was the limited availability of the plant material during some experiment meaning that experiments were not conducted in triplicate, which is desirable.

187 Recommendation for Future Research As the molecular docking indicated that plants have enzymes equivalent to microbial biodesulfurization enzymes, perhaps new plant species which have extensive sulfur metabolism such as broccoli family (Brassicae) Arabidopsis, Horseradish, onion and garlic should be tried rather than tobacco.

Hairy root culture should have a very good potential for biodesulfurization because they can be grown on minimal medium without growth regulators, they are robust and can be packed in to columns in self immobilized manner. These can then be used with repeated cycles of crude oil contact/flow and medium in order to revitalize and grow. Alternatively, immobilized plant cells in reticulate matrices or self-immobilized plant cell cultures as large aggregates in packed beds should be tried. This property of plant cell cultures in terms of ease of handling, separation from crude oil and repeated use is the best advantage of plant cell and hairy root cultures over microbial biodegradation. In the analyses of samples, better analytical methods such as improved HPLC for sugars and GC-MS for sulfur compounds and crude oil compounds should be used. Intracellular concentrations of sugars, DBT, 2-HBP and any other relevant compounds should also be measured. Intracellular sugars and sulfur compounds should be measured using not only GC- MS, HPLC but also other measurement methods such as using NMR and radio-isotope labelling (to understand the metabolic reactions involved).

Furthermore, plant metabolic reactions involved in sulfur compound degradation should be elucidated using molecular biology and genetic engineering techniques, such as proteomics, metabolomics, transcriptomics. Plant cells can be genetically modified using bacterial biodesulfurization genes combining the best attributes of the microbial and plant biocatalysts for biodesulfurization.

In order to improve the reproducibility of experimental results, history of cultures and subcultures should be kept carefully in order to track any reasons for unexpected behavior. Once an experimental protocol is developed, all the experiments should be performed following the exact protocol.

188 It was calculated in this research that contribution of DBT and even 2-HBP from crude oil can be so much higher that the ranges of DBT addition used (100 – 400ppm). It is therefore recommended that the addition of DBT should be increased. This however, creates another problem relating to the solubility of DBT in aqueous media. Therefore, perhaps the best strategy will be to analyse the sulfur compounds in the crude oil as much as and as accurately as possible and use crude oil in direct contact with plant cells. Sometimes, depending on the sulfur content of the crude oil, it may be necessary to spike it with DBT. Since cells can survive being in contact with crude oil for at least 24 h, biodesulfurization experiments using crude oil with very low sulfur compounds and added DBT should be feasible.

Kinetic models are useful in designing the experiments, the equipment and the process if they can reliably predict the behavior of the cultures. The kinetic model presented in this work needs improvement by determining its parameter values from experiments such as growth on glucose only, fructose only and combination of glucose and fructose at different levels and initial rate experiments with these sugars. Sucrose hydrolysis to glucose and fructose can be modelled with more accuracy using plant cell wall sucrose isomerase enzyme to obtain enzyme reaction parameters. Kinetic model and experimental findings should also be used for the preliminary economic assessment of biodesulfurization based on plant cell systems.

This research proved that plant cell cultures can be an alternative system for biodesulfurization for crude oil. It is therefore recommended that further research should be performed using the suggestions mentioned above.

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197 Appendix 1 Example of MATLAB Code of Kinetic Model

% Kinetic model of wild type tobacco cell suspension cultures MS only

% Parameters Vmax=0.15; Ks=13; Ygs=0.526; Yfs=0.526; Mmax1=0.006; Mmax2=0.003; Kg=3.104; Kf=8; Yxg=0.8; Yxf=0.8; Kig=1000000;

% Inputs a=288; % Time S0=23; % Initial Concentration of Sucrose G0=0; % Initial Concentration of Glucose F0=0; % Initial Concentration of Fructose X0=2.54; % Initial Concentration of Biomass

% Phase 1 f = @(t,y) [-Vmax*y(1)/(Ks+y(1))*y(4); Ygs*[Vmax*y(1)/(Ks+y(1))*y(4)]-[[Mmax1*y(2)/(Kg+y(2))]*1/Yxg]-Ms*y(4); Yfs*[Vmax*y(1)/(Ks+y(1))*y(4)]-[[Mmax1*y(3)/(Kf+y(3))]*1/Yxf]-Ms*y(4); Mmax1*[y(2)/(Kg+y(2))+y(3)/(Kf+y(3))]*y(4)]; tspan = [0, a]; yinit = [S0 G0 F0 X0]; opts = odeset('NonNegative', 1:4); [t, y] = ode45(f, tspan, yinit,opts); figure(102) plot(t,y(:,1),'r','LineWidth',3); xlabel('t'), ylabel('C'); hold on plot(t,y(:,2),'k','LineWidth',3); xlabel('t'), ylabel('C'); hold on plot(t,y(:,3),'g','LineWidth',3); xlabel('t'), ylabel('C'); hold on plot(t,y(:,4),'b','LineWidth',3); xlabel('t'), ylabel('C'); hold on

% Phase 2 f = @(t,y) [0; -[[Mmax2*y(2)/(Kg+y(2))*y(4)]*1/Yxg]-Ms*y(4); -[[Mmax2*y(3)/(Kf*(1+y(2)/Kig)+y(3))*y(4)]*1/Yxf]-Ms*y(4); Mmax2*[y(2)/(Kg+y(2))+y(3)/(Kf*(1+y(2)/Kig)+y(3))]*y(4)]; tspan = [a,504]; [rows,cols]=size(y);

198 [b, c]=find(y(:,1)<=0.1); d=t(b,c); opts = odeset('NonNegative', 1:4); fprintf('Sucrose Finished at %s hours.\n',d(1,1)); yinit = [0 y(rows,2) y(rows,3) y(rows,4)]; [t, y] = ode45(f, tspan, yinit, opts);

x = [0; 2; 72; 144; 216; 288; 360; 432; 504]; Measure_b = [2.54; 4; 7.4; 6.5; 10; 12.7; 13; 19.6; 18.5]; % Biomass Measure_r = [33; 25.8; 7.6; 1.2; 0.4; 0.7; 0.5; 0.2; 0.2 ];% sucrose Measure_g = [0; 2.9; 2.14; 2.05; 2.6; 1.05; 1.06; 1.06; 1.05];%Fructose Measure_k = [0; 10.15; 5.12; 4.13; 4.85; 0.015; 0.015; 0.06; 0.06];% Glucose

%Model result plot(t,y(:,1),'r','LineWidth',3); hold on plot(t,y(:,2),'k','LineWidth',3); hold on plot(t,y(:,3),'g','LineWidth',3); hold on plot(t,y(:,4),'b','LineWidth',3); xlabel('Time [hr]'), ylabel('Biomass and Sugar Concentration [g L^{-1}]'); % Experimental data set plot(x, Measure_b,'bs','LineWidth',1.5,'MarkerSize', 6); hold on plot(x, Measure_r,'r*','LineWidth',1.5,'MarkerSize', 6); hold on plot(x, Measure_g,'gd','LineWidth',1.5,'MarkerSize', 6); hold on plot(x, Measure_k,'kx','LineWidth',1.5,'MarkerSize', 6); set(gca, 'FontSize', 18, 'LineWidth', 1.2, 'TickDir', 'out') box on set(gcf, 'units', 'normalized', 'outerposition', [0.2 0.125 0.5 0.75]) %title('Kinetic') h(1) = plot(t,y(:,1),'r','LineWidth',1.5); h(2) = plot(t,y(:,2),'k','LineWidth',1.5); h(3) = plot(t,y(:,3),'g','LineWidth',1.5); h(4) = plot(t,y(:,4),'b','LineWidth',1.5); h(5) = plot(x, Measure_r,'r*','LineWidth',1.5,'MarkerSize', 6); h(6) = plot(x, Measure_k,'kx','LineWidth',1.5,'MarkerSize', 6); h(7) = plot(x, Measure_g,'gd','LineWidth',1.5,'MarkerSize', 6); h(8) = plot(x, Measure_b,'bs','LineWidth',1.5,'MarkerSize', 6); legend(h, 'Sucrose Model','Glucose Model','Fructose Model','Biomass Model', ... 'Sucrose', 'Glucose', 'Fructose', 'Biomass'); set(legend,'FontSize',13,'EdgeColor',[1 1 1]); clc; clear; close all;

199