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Microbial Reclamation of Alkaline Sodic Soils

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Microbial Reclamation of Alkaline Sodic Soils o à6"ì' IN Microbial Reclamation of Alkaline Sodic Soils Thesis submitted for the degree of I)octor of PhilosoPhY at the Universrty of Adelaide bY Simon Paul Odelt Deparbnent of Soil and Water Waite Agricultural Institute Glen Osmond, South Australia 2000 1l TABLE OF CONTENTS Page ABSTRACT x STATEMENT xiv ACKNOWLEDGEMENTS xv LIST OF'FIGT]RES xvi LIST OF TABLES xxi ABBREVIATIONS xxiv CHAPTER 1. Introduction I CHAPTER 2. Literature Review 2.1 Classification and formation of sodic soils 4 2.1.1 Classification of sodic soils 4 2.1.2 Natural formation of sodic soils 5 2.1.3 AnthroPogenic sodic soils 5 2.1.3.I Indiscriminate clearing and over-grazrng of native vegetation 5 2.I.3.2 Inigation of crops with poor quahty water 6 2.2 Chemical reclamation of alkaline sodic soils 6 2.2.1 Sodic soil and gYPs 6 2.2.2 Inigation 8 2.2.3 Iron PYrite I 10 2.2.4 Water soluble PolYmers 2.2.5 Heating 11 2.3 Biological reclamation of alkaline sodic soils l1 2.3.1 Organic matter and the biological reclamation of sodic soil 1l 2.3.2 Limitations of organic matter treatment t4 2.3.3 Types of organic matter t4 2.3.4 Farmyard manure mixtures t4 2.3.5 Acid cheese whey l6 2.3.6 Sewage sludge t7 2.3.7 Straw t9 2.3.8 Crops and other plants 20 2.3.9 Bagasse 22 2.3.10 Molasses 23 2.3.11 Green waste 24 2.4 Microbiology of sodic soils 24 2.4.1 Effect of alkaline conditions and NaCl on the microflora of sodic soils 24 2.4.2 Effect of soil moisture content on the microflora of sodic soils 25 2.4.3 Effect of gypsum on the microflora of sodic soils 26 2.4.4 Effect of green manure on the microflora of sodic soils 26 2.4.5 Effect of glucose on the microflora of sodic soils 27 2.5 Methods for studying the microbial populations within soils 27 2.5.t Molecular techniques 28 2.5.2 BIOLOG 29 2.5.3 Immunological methods 29 2.5.4 Fatty Acid Methyl Ester (FAME) analysis 30 2.6 Conclusion 31 ll CHApTER 3. Diversity, Alkalitolerance, Halotolerance, and Acid-Producing Capabilities of Microorganisms Cultured from an Australian Alkaline Sodic Soil 3.1 Introduction 32 3.2 Materials and Methods 33 3.2.1 Soil ProPerties 35 3.2.2 Microbiological culture media and solutions 35 3.2.3 Enumeration of non-filamentous bacteria, filamentous bacteria (actinomycetes) and frrngi 35 3.2.4 selection of non-filamentous bacteria to be used in alkali and NaCl tolerance assays 37 3.2.5 selection of filamentous bacteria (actinomycetes) to be used in preliminary alkati and Nacl tolerance assays 38 3.2.6 Selection of fungi for morphological characterisation 38 3.2.7 Alkali tolerance of filamentous and non-filamentous Watchem soil bacteria 38 3.2.8 NaCl tolerance of of filamentous and non-filamentous Watchem soil bacteria 39 3.2.9 combined alkali and Nacl tolerance of filamentous and non-filamentous \ÙVatchem soil bacteria 39 3.2.10 Assay for acid production by non-filamentous bacterial isolates 39 3.2.11 Fatry Acid Metþl Ester (FAME) identification of bacterial isolates 40 3.2.12 Identification of filamentous bacterial isolates 4l 3.2.13 Ftngal identification 4l lll 3.3 Results 42 3.3.1 Enumeration of fi lamentous bacteria (actinomycetes), non-filamentous bacteria, and fungi 42 3.3.2 Alkalitolerance and halotolerance of individual non-fi lamentous bacteria 43 3.3.2.1 Identifrcation of highly halotolerant non-filamentous bacteria by FAME analYsis 45 3.3.2.2 Identification of moderately haloalkalitolerant non-filamentous bacteria by FAME analysis 46 3.3.2.3 Identifi cation of highly haloalkalitolerant non-filamentous bacteria by FAME analYsis 47 3.3.3 Classification non-filamentous bacteria with a FAME profile similaritY of <30io 48 3.3.4 Acid production by non-filamentous bacteria: FAME identified isolates 5l 3.3.5 Acid production by non-filamentous bacteria : Unidentified isolates 52 3.3.6 Alkali and NaCl tolerance of filamentous Watchem soil bacteria (actinomycetes): Preliminary studies 54 3.3.7 Identification of filamentous bacteria (actinomycetes) 55 3.3.8 Fungal identification 57 3.4 Discussion 60 3.4.1 Alkalitolerance of the non-filamentous watchem soil bacteria 60 3.4.2 Halotolerance of the non-filamentous Watchem soil bacteria 65 lV 3.4.3 Acid production by the non-filamentous Watchem soil bacterial isolates 68 3.4.4 Alkalitolerance of filamentous bacteria (actinomycetes) isolated from the V/atchem soil: Preliminary studies 69 3.4.5 Halotolerance of filamentous bacteria (actinomycetes) isolated from the Watchem soil: Preliminary studies 70 3.4.6 Fungal identification 7T 3.5 Conclusion 73 CHAPTER 4. volatile Fatty Acid Analysis in Alkaline sodic Soils: Method Development 4.r Introduction 75 4.2 Method development 76 4.2.1 Instrumentation 76 4.2.2 Determination of chromatography conditions 77 4.2.3 Selection of the MSD analysis mode 78 4.2.4 Selection of ions to monitor 79 4.2.5 Construction of calibration curves 79 4.2.6 Selection of an internal standard 80 4.2.7 Solvent formulation 83 4.2.8 Optimisation of the extraction method 85 4.2.9 Optimum GC-MSD configuration and extraction method 87 v CHAPTER 5. Microbial Reclamation of an Älkaline Sodic Soil Using the Model Organic Substrate Glucose 5.1 Introduction 88 5.2 Materials and methods 88 5.2.1 Soil properties 89 5.2.2 Preparation of soil samples 89 5.2.3 Physical and chemical analyses 90 5.2.3.1 Soil pH and EC 90 5.2.3.2 Soluble cation analysis 90 5.2.3.3 Determination of total carbonate 9l 5.2.3.4 Determination of water soluble carbonate and bicarbonate 9l 5.2.3.5 Measurement of microbial glucose utilisation 9T s.2.3.6 Volatile fatty acid analysis of soil samples 93 5.2.4 Microbiological analyses 93 5.2.4.1 FAME analysis of soil samples 93 5.2.4.2 Methane analysis of the soil headspace 94 5.2.5 Statistical analyses 94 5.3 Results and Discussion 95 5.3.1 Microbial utilisation of glucose 95 5.3.2 Interpretation of whole soil FAME profiles 96 5.3.3 Volatile fatty acid (VFA) and FAME analysis of soil samples: W4 treatment 102 5.3.4 Volatile fatty acid (VFA) and FAME analysis of soil samples: 2W4 treatment 104 vl 5.3.s Volatile fatty acid (VFA) and FAME analysis of soil samples: 2W2treatment 108 5.3.6 Volatile fatty acid (VFA) and FAME analysis of soil samples: W2 treatment 110 s.3.7 Volatile fatty acid (VFA) and FAME analysis of soil samples:Aerobic Control t12 s.3.8 Volatile fatfy acid (VFA) and FAME analysis of soil samples:Anaerobic Control 113 5.3.9 Methane analysis of the soil headspace 115 5.3.10 Soil pH 115 5.3.11 Total carbonate/ Soluble Carbonate and Bicarbonate 118 s.3.12 Soluble Calcium 12l 5.3.13 Soluble Sodium r23 5.3.14 Soil EC 124 5.4 Conclusion t25 CHAPTER 6. Microbial Reclamation of an Alkaline Sodic Soil Using "Complex" Organic Substrates 6.1 Introduction 127 6.2 Materials and Methods 130 6.2.1 Soil properties 130 6.2.2 Ameliorant evaluation experiment 131 6.2.3 Amelioration of the Watchem soil using complex organic ameliorants 132 6.2.4 Total Carbon analYsis I32 v11 6.2.5 Interpretation of whole soil FAME profiles t34 6.3 Results and Discussion 135 6.3.1 Ameliorant evaluation experiment 135 6.3.1.1 Ameliorant evaluation experiment: Control 135 6.3.1.2 Ameliorant evaluation experiment: Wheat straw 135 6.3.1.3 Ameliorant evaluation experiment: Sheep manure r37 6.3.1.4 Ameliorant evaluation experiment: Molasses 138 6.3.1.5 Ameliorant evaluation experiment: Wheat straw and Sheep manure t39 6.3.1.6 Ameliorant screening experiment: Wheat straw and Molasses t4t 6.3.r.7 Ameliorant screening exPeriment: Super phosphate, Ammonium nitrate, Molasses and Wheat straw r42 6.3.1.8 Ameliorant screening exPeriment: Selection of ameliorants for further study r43 6.3.2 Small-scale amelioration experiment r45 6.3.2.1 Total Carbon analysis t45 6.3.2.2 Volatile Fatty Acid production and Soil FAME analysis: Control 146 6.3.2.3 Volatile Fatty Acid production and Soil FAME analysis: Molasses treatment t47 6.3.2.4 Volatile Fatty Acid production and Soil FAME analysis: Super treatment 150 v111 6.3.2.5 Soil pIV Total Carbonatel Soluble Carbonate and Bicarbonate t54 6.3.2.6 Soluble Calcium t56 6.3.2.7 Soluble Sodium 158 6.3.2.8 Soil EC 160 6.4 Conclusion 161 CHAPTER 7. General I)iscussion 7.1 Microbial populations within alkaline sodic soils 163 7.2 Microbial reclamation of alkaline sodic soils r66 REFERENCES 173 APPEI\IDIX T. Microbiological growth media and solutions 208 APPEI\DIX 2. FAME protocol for individual bacterial isolates 210 lx Abstract Alkaline sodic soils cover approximately 60% of Australia's cropping regions. These soils constitute a problem because their high pH and Na* levels cause clay swelling and dispersion, leading to a reduction in crop yields. Losses due to sodic soils are estimated at $1.4 billion per year, and remediation of these soils to increase yields would be extremely beneficial to local economies. One potential remediation method involves the stimulation of microbial populations in sodic soils to produce acid, thereby decreasing the soil pH and releasing calcium, which helps to prevent soil dispersion.
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