Isoprene degradation by soil bacteria associated with wild himalayan cherry in tropical forests thailand

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN KIM LUYEN ISOPRENE DEGRADATION BY SOIL BACTERIA ASSOCIATED WITH WILD HIMALAYAN CHERRY IN TROPICAL FORESTS – THAILAND BACHELOR THESIS Study mode : Full-time Major : Environmental Science and Management Faculty : Advanced Education Program Office Batch : 2014 - 2018 Thai Nguyen, 20/09/2018 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student name Nguyen Kim Luyen Student ID DTN1453110084 Thesis Title Supervisors Isoprene Degradation by Soil Bacteria Associated with Wild Himalayan Cherry in Tropical Forests-Thailand Dr Tho Huu Nguyen Assist Prof Thararat Chitov Supervisor’s Signature Abstract: Isoprene is a Biogenic Volatile Organic Compound (BVOC), which represents nearly one-third of total global hydrocarbon released in the atmosphere Isoprene can easily react with oxides of nitrogen to form tropospheric ozone that is harmful for life Broadleaf trees and shrubs are the greatest source of isoprene emission Certain proportion of isoprene is absorbed in soil, which can be reduced by some soil bacteria The aims of this research work are to measure isoprene degradation in the terrestrial environment, with a particular focus on soil associated with Wild Himalayan Cherry trees, one of the framework tree species used in forest restoration, and to isolate bacteria responsible for isoprene degradation Soil samples taken from underneath Wild Himalayan Cherry trees in Mae-Sa Mai restoration area were examined for their properties and rates of isoprene consumption in soil incubated with isoprene was measured using Gas Chromatography-Flame Ionization Detector (GC-FID) Bacteria responsible for isoprene consumption were isolated from soil, using minimal medium (agar and broth) supplemented with isoprene as the sole carbon source The bacterial isolates were characterised based on the features of colony and cell morphology and some biochemical tests It was found that the soil samples had the average pH of 4.4, moisture content of 40.8%, and total viable microorganisms of 5.3106 CFU/g i Consumption of isoprene by soil microorganisms was observed in the combined soil sample; which occurred at a constant rate during the first days of incubation of the soil sample with isoprene and remained unchanged afterwards Some of the soil bacterial isolates associated with Himalayan Cherry were capable of utilising isoprene as the sole carbon source Morphological and biochemical characterisation revealed these bacterial isolates to be of different types These bacterial isolates will be subjected to further identification and characterisation of their role in isoprene degradation Key words isoprene degradation, soil bacteria, Wild Himalayan Cherry Number of pages 49 Date of 24/09/2018 Submission: ii ACKNOWLEDGEMENT First of all, I would like to thank International College and The ASEAN International Mobility for Student Program for providing opportunity for me to doing my undergraduate research in Chiang Mai University I would also like to thank Microbiology Division, Department of Biology, Faculty of Science, Chiang Mai University for their technical, financial, and scientific support I am extremely grateful to my project supervisor Assistant Professor Dr Thararat Chitov at Chiang Mai University for her excellent advice and support throughout this study Being a member of your research group has been a privilege and this work would not have been possible without your immense knowledge and true guidance I am also very grateful to my internship supervisor Dr Tho Huu Nguyen at Thai Nguyen University of Agriculture and Forestry for his insightful advice, discussions and comments which brought an added value to this research work I am deeply grateful to the staff of FORRU attached to the Biology Department, Faculty of Science, Chiang Mai University for supporting me to conduct my research I thank all members of SCB 2806 laboratory especially Ms Tuangporn Uttarothai for being around to give me advice and assistance in the laboratory Thank you all for your support, friendship, and encouragement and for making the time I spent at Microbiology Division very memorable Finally, a special thank goes to my family and friends for their unequivocal support and faith in me Author Nguyen Kim Luyen iii TABLE OF CONTENT DOCUMENTATION PAGE WITH ABSTRACT i ACKNOWLEDGEMENT iii TABLE OF CONTENT iv LIST OF TABLES vi LIST OF FIGURES .vii LIST OF ABBREVIATIONS viii PART I INTRODUCTION 1.1 Research rationale 1.2 Research’s objectives 1.3 Research questions and hypothesis 1.3.1 Research questions 1.3.2 Hypothesis 1.4 Limitations 1.5 Definitions PART II LITERATURE REVIEW 2.1 Significance and Production of Isoprene 2.2 Significance of Isoprene 2.3 Isoprene’s Biological Roles 2.4 Isoprene Emission by Plants 2.5 Soil as a sink for isoprene: the effects of location, season, and isoprene degradation 2.6 Isoprene Degradation by Soil Bacteria 2.7 Isoprene-degrading Bacteria and Forest Restoration 10 PART MATERIALS AND METHODS 13 3.1 Equipment and Materials 13 3.1.1 Equipment 13 3.1.2 Microbiological Media, Reagents, and Chemicals 13 3.2 Methods 14 iv 3.2.1 Soil Sampling 14 3.2.2 Determination of soil characteristics 14 3.2.3 Determination of isoprene degradation by microorganisms in soil associated with Wild Himalayan Cherry trees 15 3.2.4 Isolation of soil bacteria capable of degrading isoprene 15 PART IV RESULTS 18 4.1 Physical and Chemical Characteristics of Soil Samples 18 4.2 Microbiological characteristics of soil samples 18 4.3 Isoprene degradation by soil microorganisms 19 4.4 Isolation of bacteria capable degrading isoprene 21 4.5 Characterisation of isoprene-degrading bacterial isolates 24 PART V DISCUSSION 27 5.1 Physical and Chemical characteristics of Soil Samples Beneath Wild Himalayan Cherry Trees 27 5.2 Microbiological Characteristics of Soil Samples 28 5.3 Investigation of Isoprene Degradation by Soil Microorganisms 29 5.4 Characterisation of Isoprene-Degrading Bacterial Isolates 29 PART VI CONCLUSION 31 REFERENCES 32 Appendix A 39 Appendix B 43 Selected Details of Analytical Procedures and results 43 Standard graph of isoprene measuring by GC-FID 43 Appendix C 48 v LIST OF TABLES Table 4.1 Location and basic characteristics of soil samples 18 Table 4.2 Count of total viable microorganisms in soil 19 Table 4.3 Isoprene consumption by soil microorganisms 20 Table 4.4 Characteristics of bacterial colony morphology 21 Table 4.5 Cell morphological characteristics of isoprene-degrading bacterial isolates 25 Table 4.6 Biochemical characteristics of isoprene-degrading bacterial isolates 26 vi LIST OF FIGURES Figure 2.1 The chemical structure of Isoprene (CH 2=C(CH2) CH=CH2 ) (Source: Polymer Science Learning Center, 2000) Figure 2.2 Isoprene cycle by sources and sinks (Source: McGenity, 2018) Figure 2.3 Wild Himalayan Cherry (Source A: Chaiudom, 2018; B, C, D: Thakur, 2015) 12 Figure 4.1 The decrease of isoprene due to consumption by soil microorganisms 20 Figure 4.2 Bacteria isolates grew on MM agar plate 22 Figure 4.3 The isolates were more turbid after 1st cultured in MM broth 23 Figure 4.4 The isolates were more turbid after 2nd cultured in MM broth 24 vii LIST OF ABBREVIATIONS Abbreviation T Explanation Temperature MC Soil moisture content CFU Colony Forming Unit MM Minimum medium G+ Gram positive G- Gram negative + Positive - Negative μl microlitre ml millilitre L litre mm millimetre pA peak area Tg teragram viii PART I INTRODUCTION 1.1 Research rationale Atmospheric pollution is currently one of the major threats to human health and the environment Due to this, it is important to gain a thorough understanding of the cycling of atmospheric pollutants Some of the atmospheric pollutants are formed from the reactions between non-pollutant compounds, such as Biogenic Volatile Organic Compounds (BVOCs) Because of their existence in large amounts, BVOCs play an important role in atmospheric chemistry, climate conditions, and air quality Isoprene (2-methyl-1,3-butadiene [C5H8]), one of the BVOCs, represents approximately one-third of total global hydrocarbon released into the atmosphere It is the second-most abundant volatile organic compound in the atmosphere after methane In areas with elevated levels of oxides of nitrogen, the oxidation of isoprene leads to the formation of tropospheric ozone, which is harmful to health and ecosystems Ozone is a major pollutant and greenhouse gas (Chameides et al., 1992), which also contribute to global warming Other effects of atmospheric isoprene oxidation include the formation of tropospheric carbon monoxide, global transport of nitrogenous compounds, extended residence times of other atmospheric trace gases, and the formation of secondary organic aerosols (Monson and Holland, 2001) The sources of atmospheric isoprene have been relatively well-studied, with terrestrial plants accounting for 90% of isoprene emissions to the atmosphere (Pacifico et al., 2009) Besides, Pegoraro (2005) reports that soil acts as a significant atmospheric sink of isoprene (3% of global emissions) Although the proportion of isoprene in soil is small, compared with that in the atmosphere, the amount is significant Since soil surface is the interface between the absorbed isoprene in soil and atmospheric isoprene, it is hope that reduction of isoprene in soil would affect the total amount of isoprene in the atmosphere Previous research works have demonstrated that some soil microorganisms possess the ability to degrade isoprene, and these organisms have association with certain types of plants Since the isoprene flux between soil and atmosphere and the interactions between plant and the associated isoprene-degrading microbes are not yet well understood, this research work was designed to address these questions Since this work was carried out in the North of MEGAN 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Decolorizer Ethanol 95% Safranin Solution Safranin O 0.25 g Distilled water 100 ml Catalase test Reagents Hydrogen peroxide 3% ml Ammonification test Reagents Nessler’s reagent Potassium iodide 7g Mercuric iodide 10 g Potassium hydroxide 10 g Distilled water 100 ml Nitrate reduction test Reagents Griess-Ilosvay’s reagents (modified) 39 Reagent Sulfanilic acid 1g Acetic acid 100 ml Reagent α-Naphthylamine 1g Ethanol 95% 100 ml Media 1.Minimum Medium Agar 5x Ca, Mg, Na, N stock 200 ml 10x Phosphate stock 100ml 1000x Trace elements stock ml 100x Vitamin solution stock 10 ml Cycloheximide (10mg ml-1) ml Agar 20 g Sterile deionised water 688 ml Constructing the medium 5x Ca, Mg, Na, N stock Sodium Chloride 5g Magnesium Sulfate Heptahydrate 5g Calcium Chloride Anhydrous 1g Ammonium Nitrate 10 g  Adjust pH to with 1M NaOH  Autoclave them in aliquots of 200 ml to allow salts to dissolve 10x Phosphate stock Sodium Hydrogen Phosphate Anhydrous 11.1 g Potassium Dihydrogen Phosphate 2.5 g  Adjust pH to with 1M HCl  Autoclave them in aliquots of 100 ml 40 1000x Trace elements stock Iron (II) Sulfate Heptahydrate 10 g Disodium EDTA 0.64 g Zinc Chloride 0.1 g Boric acid 0.015 g Cobalt (II) Chloride 0.175 g Sodium Molybdate Dihydrate 0.15 g Manganese (II) Chloride 0.02 g Nickel (II) Chloride Hexahydrate 0.01 g  Autoclave 100x Vitamin solution stock p-Aminobenzoic acid mg Folic acid mg Biotin mg Nicotinic acid mg Calcium pantothenate mg Riboflavin mg Thiamine HCL mg Pyrodoxine HCL (B6) 10 mg Cyanocobalamin (B12) 0.1 mg Thioctic acid (Lipoic acid) mg  use sterile water and glassware  adjust the pH with NaOH to  filter sterile  store in a cool and dark place  addition to medium: 10 ml/l Cycloheximide (10mg ml-1) Cycloheximide 0.1 g Sterile dH2O 10 ml 41  use sterile water and plasticware  adjust the pH with NaOH to  filter sterile and store at -20°C 2.Minimum Medium Broth 5x Ca, Mg, Na, N stock 200 ml 10x Phosphate stock 100ml 1000x Trace elements stock ml 100x Vitamin solution stock 10 ml Cycloheximide (10mg ml-1) ml Sterile deionized water 688 ml 3.Urea Agar Peptone 1g Glucose 1g Sodium chloride 5g Disodium phosphate 1.2 g Potassium dihydrogen phosphate 0.8 g Phenol red 0.012 g Sterile urea solution 50 ml Agar 15 g Distilled water 950 ml 4.Nitrate broth Potassium nitrate 1g Beef extract 10 g Peptone 10 g Sodium chloride 5g Water 1000 ml 5.Peptone water Peptone 5g Water 1000 ml 42 Appendix B Selected Details of Analytical Procedures and results Standard graph of isoprene measuring by GC-FID 3000 2500 Area 2000 1500 1000 y = 23.688x - 166.02 R² = 0.9782 500 0 20 40 60 80 100 µl * y: peak area in unit of pA x: the amount of isoprene in unit of µl µl Area 0 20 20 20 40 40 40 60 60 60 80 80 80 100 100 100 0 237.656 258.218 264.748 673.092 671.251 647.998 1122.575 1119.841 1115.166 1700.388 1732.119 1787.111 2382.766 2225.83 2391.789 y = 23.688x - 166.02 43 1.Soil moisture content Sample W1 (g) W2 (g) W3 (g) MC (%) A 0.47 5.39 3.94 41.79 B 0.56 5.6 4.11 41.97 C 0.45 5.46 4.08 38.02 D 0.43 5.47 4.08 38.08 E 0.47 5.53 4.04 41.74 F 0.51 5.5 3.99 43.39 Where: W1 = Weight of tin (g) W2 = Weight of moist soil + tin (g) W3 = Weight of dried soil + tin (g) 2.Biochemical characteristics a) Catalase test A11 A1 A3 A18 A9 A5 The appearance of gas bubbles on glass slide 44 b) Ammonification test A11 A3 A18 A16 A9 A5 The colour changed from white to brown for isolates A3, A5 and A11 c) Nitrate reduction test A18 A9 A11 A16 A3 A5 The colour changed from white to red in isolates A3, A5 and A11 45 A18 A9 A16 Zn powder was added onto A9, A16, A18 and resulted in development of red coloured substance d) Urease test There was no change in colour of all plates contained isolates 46 Incubated agar plates in anaerobic jars contained isoprene inside under room temperature Colonies grew after spread plating Incubated vials under shaking condition at room temperature (~25°C) for days 47 Appendix C Pictures of author’s activities Collecting soil samples Placing Minimum Medium Agar onto plates Isolation of soil bacteria using spread plating 48 Profile Name Nguyen Kim Luyen Date of birth May 5, 1996 University Thai Nguyen University of Agriculture and Forestry, Vietnam Telephone +84965051996 e-mail nguyenkimluyen1996@gmail.com 49 ... isoprene- degrading bacteria in soil? 1.3.2 Hypothesis Bacteria in soil associated with Wild Himalayan Cherry trees in tropical forest could absorb isoprene 1.4 Limitations Limited time is the main culprit... 3.2.3 Determination of isoprene degradation by microorganisms in soil associated with Wild Himalayan Cherry trees The rates of isoprene degradation by soil microorganisms were measured using Gas... Supervisors Isoprene Degradation by Soil Bacteria Associated with Wild Himalayan Cherry in Tropical Forests- Thailand Dr Tho Huu Nguyen Assist Prof Thararat Chitov Supervisor’s Signature Abstract: Isoprene