UNIVERSITY OF PADOVA DOCTORATE SCHOOL OF CROP SCIENCE CURRICULUM AGROBIOTECHNOLOGY - CYCLE XXIII Department of Agricultural Biotechnology Microbial biodiversity in a wooded riparian zone specifically designed for enhancing denitrification process Director of the school: Prof. Andrea Battisti Supervisor: Prof. Sergio Casella Co-supervisor: Prof. Marina Basaglia PhD student: Md. Mizanur Rahman THESIS SUBMISSION DATE January 31st, 2011 ii Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgment has been made in the text. Md. Mizanur Rahman January 31st, 2011 A copy of the thesis will be available at http://paduaresearch.cab.unipd.it/ iii iv Dedicated To My late Father and family. v vi INDEX LIST OF ABBREVIATION............................................................................................................. 2 ABSTRACT....................................................................................................................................... 6 RIASSUNTO ..................................................................................................................................... 7 1. INTRODUCTION....................................................................................................................... 10 1.1. The wooded riparian zone ....................................................................................................... 11 1.1.1. Functions of the riparian buffer zones................................................................................. 13 1.1.2. Modes of particulate and dissolved nutrient transport in the riparian buffer zone.............. 14 Surface runoff................................................................................................................................ 15 Subsurface flow and drainage ....................................................................................................... 15 1.2. Nitrogen removal in riparian buffers ..................................................................................... 16 1.2.1. Mechanisms of nitrogen removal........................................................................................ 17 1.2.1.1. Ammonification ......................................................................................................... 17 1.2.1.2. Nitrification................................................................................................................ 17 1.2.1.3. Denitrification............................................................................................................ 18 1.2.1.4. Plant uptake................................................................................................................ 21 1.2.1.5. Physicochemical processes ........................................................................................ 21 1.2.1.5a. Ammonia adsorption........................................................................................... 21 1.2.1.5b. Sedimentation ..................................................................................................... 22 1.2.2. Environmental factors affecting nitrogen removal efficiency............................................. 22 1.2.2a. Temperature affecting nitrogen removal efficiency .......................................................... 22 1.2.2b. Hydraulic residence time affecting nitrogen removal efficiency ...................................... 23 1.2.2c. Types of vegetation affecting nitrogen removal efficiency............................................... 23 1.3. Biodiversity in soil .................................................................................................................... 24 1.3.1. Microbial diversity in soil..................................................................................................... 25 1.3.1.1. Bacterial diversity in soil .....................................................................................................26 1.3.1.1a. Proteobacteria.................................................................................................................27 1.3.1.1b. Acidobacteria.................................................................................................................28 1.3.1.1c. Verrucomicrobia............................................................................................................28 1.3.1.1d. Cytophagales..................................................................................................................28 1.3.1.1e. Actinobacteria ................................................................................................................28 1.3.1.1f. Firmicutes........................................................................................................................29 1.3.1.1g. Planctomycetes ..............................................................................................................29 1.3.1.2. Effect of different factors on soil microbial diversity......................................................29 1.3.1.2.1. Soil type as the determinant of the structure of microbial communities in soil..29 1.3.1.2.2 Plant type as the determinant of the structure of microbial communities in soil .30 1.3.1.2.3. Agricultural management regime as the determinant of the structure of microbial communities in soil. ......................................................................................................................33 1.3.1.2.3a. Cropping effect ........................................................................................... 34 1.3.1.2.3b. Soil amendment and tillage ........................................................................ 35 1.3.1.2.4. The soil habitat .............................................................................................................35 1.3.1.2.4a. The effect of soil structure and environmental conditions on microbial diversity.....................................................................................................................................36 1.3.1.3. Microbial diversity versus community structure ..............................................................36 1.3.1.3.1. Definition and measurement of microbial diversity................................................37 1.3.1.3.1.1 Diversity index......................................................................................................37 1.3.1.4. Methods for the assessment of soil microbial diversity...................................................38 vii 1.3.1.4.1. Cultivation – based methods.......................................................................................38 1.3.1.4.2. Cultivation- independent methods.............................................................................39 1.3.1.4.2.1. Fatty acid methyl ester (FAME) analysis ........................................................39 1.3.1.4.2.2. Metabolically active communities (Direct methods) .....................................40 1.3.1.4.2.3. DNA based methods ...........................................................................................40 1.3.1.4.2.3a. Clone libraries..................................................................................... 41 1.3.1.4.2.3b. Microbial community fingerprinting techniques................................ 41 1.3.1.5. Future perspectives ...............................................................................................................45 2. EXPERIMENTAL SITE ............................................................................................................47 2.1. Introduction.............................................................................................................................. 48 2.2. The study area .......................................................................................................................... 48 2.3. Experimental site description.................................................................................................. 50 2.3.1. Soil characteristics............................................................................................................... 52 2.3.2. Site hydrology ..................................................................................................................... 52 2.3.3. Vegetation ........................................................................................................................... 53 3. OBJECTIVES ............................................................................................................................. 55 3.1. General objectives .................................................................................................................... 56 3.2. Specific objectives..................................................................................................................... 56 4. METABOLIC ACTIVITY AND MICROBIAL DIVERSITY OF THE WOODED RIPARIAN STRIP SOIL BY USING CULTURE DEPENDENT AND INDEPENDENT METHODS. ..................................................................................................................................... 57 4.1. Introduction.............................................................................................................................