DOCTOR OF PHILOSOPHY Effect of soil amendments on the chemical transformation of arsenic and the microbial diversity in rice paddy microbiome Begum, Monira Award date: 2021 Awarding institution: Queen's University Belfast Link to publication Terms of use All those accessing thesis content in Queen’s University Belfast Research Portal are subject to the following terms and conditions of use • Copyright is subject to the Copyright, Designs and Patent Act 1988, or as modified by any successor legislation • Copyright and moral rights for thesis content are retained by the author and/or other copyright owners • A copy of a thesis may be downloaded for personal non-commercial research/study without the need for permission or charge • Distribution or reproduction of thesis content in any format is not permitted without the permission of the copyright holder • When citing this work, full bibliographic details should be supplied, including the author, title, awarding institution and date of thesis Take down policy A thesis can be removed from the Research Portal if there has been a breach of copyright, or a similarly robust reason. 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Sep. 2021 Abstracts Effect of soil amendments on the chemical transformation of arsenic and the microbial diversity in rice paddy microbiome A thesis submitted to the School of Biological Sciences Queens University Belfast For the degree of Doctor of Philosophy Monira Begum Summer, 2021 i Abstracts Abstracts Paddy management practices greatly influence arsenic biogeochemistry. For sustainable rice production, rice needs to be free from arsenic. Different soil amendments such as animal manure in flooded paddy soil could enhance arsenic reductive mobilization, biomethylation, and volatilization which could be a useful option for the arsenic detoxification process. Also, arsM genes in composting manure could enhance arsenic methylation. Furthermore, cattle manure also enhances arsenic mobilization and increased arsenic assimilation in rice grain in some regions. However, the application of biomass ash, a source of silicon (Si) could be beneficial for rice growth, as silicate competes with arsenite for root uptake and hence has the potential to decrease translocation of arsenic into the rice grain. In contrast, the application of chemical fertilizer is not sustainable and could lead to long-run problems. As such, it is hypothesized “do different fertilizers affect the rice paddy microbiome and influence arsenic biogeochemistry, arsenic speciation, methylation and ultimately assimilation of arsenic into rice grain?” Thus, the objectives of this research were to ascertain the effect of different soil amendments on arsenic methylation and mobility in paddy soil and rice arsenic accumulation over time. In addition, how soil amendments affect the abundance and diversity of the arsM, 16S rRNA bacterial and ITS1 fungal genes in the rice microbiome was also investigated. Therefore, the research approach was to set up a rice microcosm experiment in a growth cabinet with a rice cultivar, ‘Yongyou12’ (a Japonica indica hybrid) with application of five different treatments- mineral fertilizers, full- slurry, half-slurry, ash and no application (four replicates per treatment), were applied to study the arsenic biogeochemistry and microbial population in rice paddy microbiome. Rice soil porewater chemical characterization at 7, 14, 28, 56, 84 d, during heading (99-126 d), grain fill (107-134 d) and dry stage (145-170 d) showed a highly significant effect of time on overall soil and arsenic biogeochemistry, while different soil treatments resulted in a significant response only for pH, Eh, Asi, B, Zn, Rb, Pb. Rice porewater arsenic species (Asi and MMA, DMA and TMAO) showed a strong correlation with different porewater properties, B, P, Mn, Fe, Co, Cu, Zn, Se, Rb, Sr, Mo, Cd and Pb. In rice shoot, overall treatments had a significant response ii Abstracts for B, P, Cr, Mn, Fe, Co, Ni, Zn, Rb, Cd, S, K, Na and Sn; while in rice grain, the effects were not pronounced. The 16SrRNA and arsM bacterial abundance in rice rhizosphere soil at 14 d, 56 d, grain fill (107-134 d) and dry stage (145-170 d) revealed a significant increase in both arsM and 16S rRNA gene copy number over time. The highest bacterial abundance was observed during the grain fill (107-134 d) compared to other time points. For treatments, a significant increase in 16S rRNA gene copy number was identified in response to the full slurry treatment. Amplicon sequencing at the grain fill stage of both the bacterial 16S rRNA and fungal ITS1 genes showed that diversity significantly varied among the three compartments, rice rhizosphere soil, root and shoot. Soil showed the highest level of richness and diversity, followed by the rice root, while a low level of richness and diversity was seen in the shoot. The most pronounced treatment effects on microbial operational taxonomic units (OTU’s) were observed for soil bacterial OTU’s. In most instances, copy number was increased in response to full-slurry and/or half-slurry treatment or decreased in response to ash, full-slurry and/or half- slurry, while mineral fertiliser had no or little effect. For bacterial OTU’s, Clostridium was the most relatively abundant genus in rhizosphere soil and shoot and Geobacter was the most relatively abundant in rice root. Geobacter, Clostridium, Anaeromyxobacter, Opitutus, Desulfocapsa, Methylocystis and Gemmatimonas were relatively abundant in all three compartments. Some of these have arsenic detoxification and methylation and could control the N, C and Fe cycling. The statistical analysis identified a significant increase in response to the manure treatment for several genus-level bacterial OTU’s in soil (Sarcina, unclassified genera within the phylum Cyanobacteria, Bacteroidetes, the order Clostridiales and the family Rhodothermaceae). These could be involved in arsenic methylation and volatilization as found in the literature. For fungal ITS1, Ascomycota annotated OTU’s was identified as the most abundant phylum in all three compartments. Pseudeurotium, Talaromyces, Cladosporium and Mortierella were relatively abundant genus in rhizosphere soil, Cladosporium, Fusarium, Pseudeurotium, Sarocladium, Talaromyces and Zopfiella in root and Cladosporium, Penicillium, Pseudeurotium in the shoot. Statistical analysis identified only a small number of fungal OTU’s with significant treatment effect. iii Abstracts Application of full-slurry, half-slurry, and ash showed much stronger effect compared to the chemical fertilizer in controlling rice arsenic chemistry and could be applied after filed trial. It is clear that the manure, ash, and half-slurry had some significant effect and could be beneficial to increase arsenic methylating bacteria and fungi in rice microbiome. iv Table of contents Table of Contents Page no. Chapter 1 Introduction………………………………………………………………………….. 1 1.1 Arsenic (As) a potential problem in rice environment.……………………… 1 1.2 Sources and forms of Arsenic……………………………………………………………. 2 1.3 The biogeochemistry of arsenic in paddy soil……………………………………. 3 1.3.1 Flooding induced arsenic mobilization…………………………………………… 3 1.3.2 Arsenate reduction………………………………………………………………………… 4 1.3.3 Arsenite oxidation…………………………………………………………………………. 5 1.3.4 Arsenic methylation, volatilisation and demethylation………………….. 5 1.4 Arsenic uptake pathways in rice ………………………………………………………. 6 1.4.1 Silicic acid transporters………………………………………………………………….. 6 1.4.2 Phosphate transporters…………………………………………………………………. 8 1.5 Microbes and their role in arsenic dynamics in paddy soil………………… 8 1.6 Microbial interaction and function in paddy soil………………………………. 10 1.7 Effects of soil amendments………………………………………………………………. 11 1.8 Thesis objectives and hypothesis……………………………………………………… 13 1.9 Outline of the thesis and chapters……………………………………………………. 14 Chapter 2 Rice microcosms……………………………………………………………………. 15 2.1 Introduction……………………………………………………………………………………… 15 2.2 Collection of soil for the experiment………………………………………………… 16 2.2.1 Location and topography of sampling……………………………………………. 16 2.3 Experimental design…………………………………………………………………………. 17 2.4 Collection and storage of samples……………………………………………………. 23 Chapter 3 Impact of soil management and time on arsenic biogeochemical cycling of paddy soils…………………………………………………… 29 3.1 Introduction……………………………………………………………………………………… 29 3.2 Materials and methods…………………………………………………………………….. 32 3.2.1 Porewater characterisation…………………………………………………………… 32 3.2.1.1 Determination of pH and Eh……………………………………………………….. 32 3.2.1.2 Porewater processing…………………………………………………………………. 33 3.2.1.3 Determination of Total Organic Carbon (TOC)…………………………….. 33 v Table of contents 3.2.1.4 Speciation of arsenic in rice porewater ……………………………………… 33 3.2.1.5 Total elemental analysis of rice porewater……………….................... 34 3.2.2 Rice biomass information……………………………………………………………….