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Bacterial Diversity and Functions in Soils Amended with Biochar and Organic Fertilizers Bacterial diversity and functions in soils amended with biochar and organic fertilizers Jun Ye A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science December 2016 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Ye First name: Jun Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Biotechnology anD Biomolecular Sciences Faculty: Science Title: Bacterial diversity anD functions in soils amenDeD with biochar anD organic fertilizers Abstract 350 worDs maximum: Organic farming relies on the activities of bacterial communities for optimal soil productivity. Understanding the responses of bacterial communities to different soil amendments, including organic fertilizers and biochars, can provide information for soil management. This thesis firstly describes the bacterial diversity and functions that are central to soil processes of organic soils and their responses to fertilizers with different C/N ratios. Secondly, this thesis analyzes the effect of a mineral-enhanced biochar (MEB) on the bacterial community of organically amended soil. Finally, the thesis investigates the direct interactions between biochar and bacteria that underpin metabolic processes in the soil. Bacterial taxa that are resilient to different fertilizers were identified and defined as the core community of organic soil. The phyla Bacteroidetes and Planctomycetes, the family Cytophagaceae and the class Acidobacteria-5 were thus found in organic soil regardless of the type of fertilizer being applied. These core bacterial taxa were further linked to the functional potential of organic soil. The C/N ratio of fertilizer was also found to have a positive correlation with microbial N assimilation in organic soil. MEB was applied in combination with compost to soil and this resulted in synergistic effects on soil properties. Specifically, the soil nitrate content was increased, which correlated with an enrichment of bacterial nitrifiers due to the MEB addition. As a consequence, plants produced larger leaves, which demonstrates that MEB could be used to manipulate specific agricultural outcomes in organic farming. To understand the detailed mechanism that supports the beneficial effects of biochar and MEB, a novel method was developed to visualize the in situ interactions between bacteria and surfaces on a single-cell level. Distinct bacterial communities were found to exist on the surface of biochar and MEBs compared to surrounding soils and surface-associated bacteria were found to have the capacities to fix carbon dioxide using chemolithotrophic processes. This provides a bacterial mechanism on how biochar and MEBs can drive carbon sequestration into the soil environment. Together, the discoveries and models presented in this thesis provide new insights into the functions of soil microbiomes in organically amended soil. Declaration relating to DisPosition of Project thesis/Dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). 25/08/2016 ………………………………………… ……………………………………..………… ……….……………………...…… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: Abstract Organic farming relies on the activities of bacterial communities for optimal soil productivity. Understanding the responses of bacterial communities to different soil amendments, including organic fertilizers and biochars, can provide information for soil management. This thesis firstly describes the bacterial diversity and functions that are central to soil processes of organically managed soils (organic soils) and their responses to fertilizers with different C/N ratios. Secondly, this thesis analyzes the effect of a mineral-enhanced biochar (MEB) on the bacterial community of organically amended soil. Finally, the thesis investigates the direct interactions between biochar and bacteria that underpin metabolic processes in the soil. Bacterial taxa that are resilient to different fertilizers were identified and defined as the core community of organic soil. The phyla Bacteroidetes and Planctomycetes, the family Cytophagaceae and the class Acidobacteria-5 were thus found in organic soil regardless of the type of fertilizer being applied. These core bacterial taxa were further linked to the functional potential of organic soil. The C/N ratio of fertilizer was also found to have a positive correlation with microbial N assimilation in organic soil. MEB was applied in combination with compost to soil and this resulted in synergistic effects on soil properties. Specifically the soil nitrate content was increased, which correlated with an enrichment of bacterial nitrifiers due to the MEB addition. As a consequence, plants produced larger leaves, which demonstrates that MEB could be used to manipulate specific agricultural outcomes in organic farming. To understand the detailed mechanism that supports the beneficial effects of biochar and MEB, a novel method was developed to visualize the in situ interactions between bacteria and surfaces on a single-cell level. Distinct bacterial communities were found to exist on the surface of biochar and MEBs compared to surrounding soils and surface- associated bacteria were found to have the capacities to fix carbon dioxide using chemolithotrophic processes. This provides a bacterial mechanism on how biochar and MEBs can drive carbon sequestration into the soil environment. i Together, the discoveries and models presented in this thesis provide new insights into the functions of soil microbiomes in organically amended soil. ii Acknowledgements First and foremost, I would like to sincerely thank my supervisor A/Prof. Torsten Thomas for his encouragement when I encountered with personal issues during the earliest days of my study, for his patience when I need to practice my English skills, for his motivation when I had difficulties to proceed on the rough road of experiments. There will be a very long list if I must write down all things that indebted and learned form Torsten. I should say that he has taught me so much beyond the boundaries of scientific knowledge. He is always there when I seek for guidance. Truly, he is more like a best friend of mine. Secondly, I deeply appreciate the support of Dr. Shaun Nielsen, who generously shared his time, knowledge and imaginations, enlightening me to gain a rigorous sense for statistical analysis and R language. It is my pleasure to work with him over the four years. I would also like to thank all cooperators who made my PhD study fruitful. Thanks to Prof. Stephen Joseph from the School of Materials Science and Engineering in UNSW for his in-depth understanding about biochar and for his insistence that made the observation of in situ iron oxidization on biochar possible. Thanks to Prof. Danfeng Huang from Shanghai Jiao Tong University for her continuous support since my Master study in China. Thanks members from Danfeng’s group, including Dr. Pablo Gonzalez Perez, Miss Rui Zhang and Yun Liang, for helping me to set up the experiments and collect data on organic soils. Those electron microscopic images in this thesis would not have been presented without the technical expertise of the people from the Electron Microscope Unit in UNSW. Especially, many thanks to Dr. Jenny Norman for training me on all sorts of relevant skills, to Dr. Simon Hager for replacing specimens from JEOL 7001F field emission scanning electron microscopy every time. My thanks also to Dr. Bill Gong from the Solid State and Elemental Analysis Unit for doing X-ray photoelectron spectroscopy on biochar, to Sydney Liu Lau from the Bioanalytical Mass Spectrometry Facility for assisting the analysis of nanogold labelled oligonucleotide. iii Dozens of people at the Centre for Marine Bio-Innovation have immensely helped and taught me. Thanks to Esra Kurnaz for all the administrative assistance. I wish all the best for her new job. Thanks to Mansour Baligh for all his advice when I was frustrated at the beginning of my study. I wish him every success in the future. I would particularly like to thank the following people for their support and friendship, Galaxy Qiu, Tamsin Peters, Michael Liu, Lu Fan, Vipra Kumar, Harry Siang, Rajesh T, Willis Song, David Reynolds, Mary Nguyen, Cristina Diez-Vives, Ana Dos Santos Esteves. Special thanks to Yan Liao, Mukan Ji, Tammy Tang, Tianzhe Liu, Lu Yang, Lifu Sheng, Songzhe Fu, Huizhi Hu and Miaomiao Zhang for all the laughter and happy time at every lunch break. I will surely miss that. To my love, Amy Su, who is
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