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The Efficacy of Plants to Remediate Indoor Volatile Organic Compounds and the Role of the Plant Rhizosphere During Phytoremediation

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The Efficacy of Plants to Remediate Indoor Volatile Organic Compounds and the Role of the Plant Rhizosphere During Phytoremediation THE EFFICACY OF PLANTS TO REMEDIATE INDOOR VOLATILE ORGANIC COMPOUNDS AND THE ROLE OF THE PLANT RHIZOSPHERE DURING PHYTOREMEDIATION Manoja Dilhani De Silva Delgoda Mudiyanselage Staffordshire University A thesis submitted in partial fulfilment of the requirement of Staffordshire University for the degree of Doctor of Philosophy January 2019 This work is dedicated to my son. You have made me stronger, better and more fulfilled than I could have ever imagined. I love you to the moon and back. “There's so much pollution in the air now that if it weren't for our lungs there'd be no place to put it all” - Robert Orben This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. Abstract A wide range of volatile organic compounds (VOC) are released from building materials, household products and human activities. These have the potential to reduce indoor air quality (IAQ), poor IAQ remains a serious threat to human health. Whilst the ability of the single plant species to remove VOC from the air through a process called phytoremediation is widely recognised, little evidence is available for the value of mixed plant species (i.e. plant communities) in this respect. The work reported herein explored the potential of plant communities to remove the three most dominant VOCs: benzene, toluene and m-xylene (BTX) from indoor air. During phytoremediation, bacteria in the root zone (rhizosphere) of plants are considered the principal site contributing to the VOC reduction. This project explored BTX degrading bacteria in the rhizosphere through culture-dependent and independent approaches. This research revealed that mixed plant culture could remove low and high concentrations of BTX from the air. Interestingly, 100 ppm BTX removal rates by single plant species were higher than the removal rate observed for mixed plant species in all cases. At the low concentration (10 ppm), all plants showed higher removal rates of benzene and toluene than m-xylene. Some bacteria in the rhizosphere utilised gaseous BTX as their sole carbon and energy sources were isolated on minimal salt agar. The majority of isolated bacteria were Gram-positive and belonged to the phylum Actinobacteria. Most of the identified bacteria belonged to the genera Microbacterium, Rhodococcus, Arthrobacter and Pseudomonas. In considering the impact of BTX upon the rhizosphere microbiome, it was shown that overall there were little compositional and functional changes following exposure to 10 ppm gaseous benzene. Findings from this work enhanced our understanding of the benefit of indoor plants in relation to VOC remediation and the consequent improvement of phytoremediation systems for the protection of public health. i Acknowledgement First, I would like to thank my principal supervisor Dr. Peter C. Gowland for his support, guidance, and advice throughout my Ph.D. I would also like to thank my former principal supervisor and current external supervisor, Dr. Daniel P. Tonge for his continued support, advice and input. A special thanks is owed to Professor John Dover and Dr Alison Davidson for their advice, help and technical guidance. I would like to acknowledge the assistance and advise of Dr Julian Partridge, especially in the latter stages of the project. My sincere gratitude goes to the technical skills specialists’ team, Ian Hopkins, Dr Rob Manning, Mishele Barrigas, Caroline Newman, Denise Figgins, Rosie Duncan, Ian Pinks, Simon Cooper and Sarah Appelbee. I am thankful to my fellow research students, especially Udeshika Weerakkody (now Dr), Esme Hookway and Eleanor Atkins for their great support. I also thank all friends who endured listening to various PhD related woes and made me feel better. I owe a special thanks to my parents and brothers for their untiring support and love throughout this journey. The greatest thanks go to my dearest husband for his tremendous support and being there with me throughout this challenging time. I thank my son for providing a cheerful atmosphere at home, I appreciate his patience and support during mommy’s Ph.D. studies. Lastly but not least, I would like to thank the long list of people that I haven’t got space to thank individually for your guidance, help, advice, encouragement, enthusiastic support over these years. ii Table of contents 1.1. Introduction ............................................................................................................................. 2 1.2. Air pollution and air pollutants ................................................................................................ 5 1.3. Indoor air pollution .................................................................................................................. 7 Volatile organic compounds in indoor air ......................................................................... 7 Benzene, toluene and m-xylene (BTX) ..................................................................... 10 1.3.1.1.1. Benzene ............................................................................................................. 11 1.3.1.1.2. Toluene ............................................................................................................. 11 1.3.1.1.3. m-xylene............................................................................................................ 12 1.4. Bioremediation of VOC from the environment ..................................................................... 12 Phytoremediation of VOC by indoor plant species ......................................................... 13 S. wallisii, C. comosum and H. helix as VOC removing indoor plant species ........... 19 Mechanism involved during phytoremediation of VOC in plant leaves .................. 21 Rhizoremediation and mechanism involved during rhizoremediation ................... 23 1.5. Culture-dependent approaches to analyse bacterial community diversity .......................... 30 1.6. Culture-independent approach to analyse bacterial community diversity ........................... 31 Molecular techniques involved in community analysis .................................................. 32 iii High through-put sequencing of community DNA ......................................................... 33 Brief overview of the method involved in Illumina MiSeq sequencing .......................... 33 Bioinformatic data analysis ............................................................................................. 36 1.7. Methods employed in monitoring VOC concentrations in air samples ................................. 37 Background of Aeroqual VOC monitoring method ......................................................... 37 PID technology in the sensor ................................................................................... 39 Analysis of VOC in air using gas chromatography/flame ionisation detection ............... 41 Choice of Tenax TA as a sorbent material for VOC analysis .................................... 42 Thermal desorption ................................................................................................. 43 1.8. Aims of the project................................................................................................................. 45 Media and reagents ............................................................................................................... 47 Plant species selection ........................................................................................................... 48 Exposure of plants to VOC to investigate possible changes to the rhizosphere community 49 Rhizosphere soil sample collection ........................................................................................ 50 Total DNA extraction from rhizosphere ................................................................................. 51 QIAamp fast DNA stool mini kit ...................................................................................... 51 MO BIO PowerSoil® DNA isolation kit ............................................................................. 52 Determination of DNA concentration ............................................................................. 53 Amplification of 16S rRNA gene from rhizosphere community ............................................. 53 Agarose gel electrophoresis ................................................................................................... 54 Purification of PCR products .................................................................................................. 55 Rhizosphere bacterial community profiling using Illumina MiSeq Sequence platform ......... 55 iv Illumina MiSeq Sequencing ............................................................................................. 56 Taxonomical classification of soil bacterial community ................................................. 56 Functional classification of soil bacterial community ..................................................... 57 Isolating VOC degrading bacteria from rhizosphere soil ..................................................... 58 Characterisation and identification of bacterial isolates ..................................................... 59 Classical approach to identify bacteria ......................................................................... 59 Molecular approach to identify bacteria .....................................................................
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