Mycoremediation Protocols (Springer Protocols Handbooks)

Preface
Contents
Contributors
Chapter 1: Isolation, Enrichment, and Characterization of Fungi for the Degradation of Organic Contaminants
	1 Introduction
	2 Materials
	3 Screening of Pesticide-Degrading Fungal Strains
		3.1 Isolation of the Fungal Strains
		3.2 Purification of Fungal Isolates
		3.3 Morphological Characterization of Fungi
		3.4 Microscopic Observation of Fungi by Temporary Wet Mount Technique
		3.5 Mounting of Fungal Cells by Lactophenol Cotton  Blue
		3.6 Molecular Characterization of Fungi
			3.6.1 Genomic DNA Extraction
			3.6.2 Polymerase Chain Reaction (PCR)
	4 Notes
	References
Chapter 2: Protocol for the Assessment of Mycoremediation of Polycyclic Aromatic Hydrocarbons
	1 Introduction
	2 Materials
		2.1 PAHs Extraction and Quantification by HPLC-PDA
		2.2 Soil Enzymatic Activities
			2.2.1 Dehydrogenase Activity (DHA)
			2.2.2 Total Microbial Activity (TMA)
			2.2.3 Urease Activity (UA)
		2.3 Germinability Test
		2.4 Test of Survival and Reproduction of Collembolan (Folsomia candida)
	3 Methods
		3.1 Assessment of the PAH Mycodegradation: PAH Extraction and Quantification
		3.2 Recovery of Soil Microbial Functions
			3.2.1 Dehydrogenase Activity
			3.2.2 Total Microbial Activity
			3.2.3 Urease Activity
		3.3 Germination Test
		3.4 Test of Survival and Reproduction of a Type of Springtails/Collembolan (Folsomia candida)
	4 Notes
	References
Chapter 3: Characterization and Screening of Pesticide-Degrading Indigenous Fungi from Soil and Water
	1 Introduction
	2 Materials
		2.1 Pesticides, Reagents, and Solvents
		2.2 Media
		2.3 Soil Sampling/Water Sampling
	3 Methods
		3.1 Isolation of Pesticide-Degrading Fungi from Contaminated Soil/Water
		3.2 Identification and Characterization of Fungal Isolates
		3.3 Growth Inhibition Studies
		3.4 Pesticide Biodegradation Assay for Screening of Potential Fungal Isolate
	4 Notes
	References
Chapter 4: Mycoremediation of Synthetic Textile Dyes by Fungi Isolated from Textile Wastewater Effluent and Soil
	1 Introduction
	2 Materials
		2.1 Modified Kirk´s Medium
		2.2 Potato Dextrose Broth (PDB)
	3 Methods
		3.1 Preparation of Textile Dyes and Quantification
		3.2 Sample Collection
		3.3 Analysis of Physicochemical Parameters of the Collected Water Samples
		3.4 Enrichment of Textile Wastewater and Soil Samples
		3.5 Isolation of Textile Dye-Decolorizing Fungi
		3.6 Screening of Textile Dye-Decolorizing Fungi
			3.6.1 Solid Medium Screening
			3.6.2 Liquid Medium Screening
		3.7 Optimization of the Textile Dye Decolorization Processes by Selected Fungi
		3.8 Assessment of the Toxicity of the Decolorized Dye Solutions
	4 Notes
	References
Chapter 5: Protocol for Screening Endophytic Fungi Against Heavy Metals
	1 Introduction
	2 Materials
		2.1 Endophytic Fungus Isolation and Purification
		2.2 Heavy Metals Screening of the Fungal Isolates
	3 Methods
		3.1 Isolation of Endophytic Fungi
		3.2 Preparation of PDA Supplemented with the Heavy Metal Solution (Prior to Screening)
		3.3 Screening of Fungal Isolates against Heavy Metals
	4 Notes
	References
Chapter 6: Screening of Phyllosphere Fungi Inhabiting in Urbanized Areas for Phylloremediation Capabilities of Polyaromatic Hy...
	1 Introduction
	2 Materials
		2.1 Isolation of Phyllosphere Fungi
			2.1.1 Chemical Composition of Bacto-Bushnell Hass (BBH) Broth Medium
		2.2 Screening and Confirmation of PAH-Degrading Potential of Phyllosphere Fungi
			2.2.1 Screening of PAH Degradation Potential Using Plate Assay
			2.2.2 Determination of PAH Degradation Potential Using UV-Visible Spectroscopy
			2.2.3 HPLC Analysis of PAH Degradation
		2.3 Identification of PAH-Degrading Fungi
			2.3.1 Molecular Analysis to Identify Them up to Species Level
				Extraction of Fungal Genomic DNA
		2.4 Enzyme Assay
	3 Methods
		3.1 Leaf Sample Collection
		3.2 Isolation of Phyllosphere Fungi
			3.2.1 Isolation of Phylloplane-Inhabiting Fungi
			3.2.2 Isolation of Endophytic Phyllosphere Fungi
			3.2.3 Microscopic Observations of Leaf Endophytes
			3.2.4 Endophytic Fungal Observations Through  SEM
		3.3 Screening and Confirmation of PAH-Degrading Potential Phyllosphere Fungi
			3.3.1 Plate Assay (Primary Screening)
			3.3.2 Spectrophotometric Analysis (Secondary Screening)
			3.3.3 HPLC Quantification of Polyaromatic Hydrocarbon (Confirmation Test)
		3.4 Identification of Phyllosphere Fungi
			3.4.1 Identification of Phyllosphere Fungi up to Genus Level
			3.4.2 Molecular Identification of PAH-Degrading Phyllosphere Fungi
				Extraction of Fungal Genomic  DNA
				Amplification of Fungal 18S rRNA Region of Genomic DNA by PCR Method
				Agarose Gel Electrophoresis
				DNA Sequencing
				GenBank Search
		3.5 Enzymatic Degradation of PAH by Extracellular Enzymes
			3.5.1 Manganese-Dependent Peroxidases (MnP) Activity
			3.5.2 Lignin Peroxidases (LiPs) Activity
			3.5.3 Laccases Activity
			3.5.4 Calculation of Enzyme (MnP, LiP, and Laccases) Activities
	4 Notes
	References
Chapter 7: Methods for Design and Bioremediation Applications of Reactors Based on Immobilized Fungi
	1 Introduction
	2 Materials
		2.1 Trickle-Bed Bioreactors
			2.1.1 WRF-Based Bioreactor for Degradation of Textile  Dyes
				Scaling-Up of Trickle-Bed Reactors
			2.1.2 Spent Mushroom Substrate-Based Bioreactor for Degradation of Endocrine-Disrupting Compounds (EDCs)
			2.1.3 SMS-Based Bioreactor for Degradation of Polychlorinated Biphenyls (PCBs)
		2.2 Rotating Drum Biological Contactor (RDBC) for Biodegradation of Textile  Dyes
		2.3 Rotating Disk Reactor (RDR) for Biodegradation of Textile  Dyes
			2.3.1 Scaling-Up of Rotating Biological Contactors
	3 Methods
		3.1 White-Rot Fungus-Based Bioreactor for Degradation of Textile  Dyes
			3.1.1 Examples of Application
		3.2 SMS-Based Bioreactor for Degradation of  EDCs
			3.2.1 Examples of Application
		3.3 SMS-Based Bioreactor for Degradation of  PCBs
			3.3.1 Examples of Application
		3.4 Rotating Drum Biological Contactor (RDBC) for Biodegradation of Textile  Dyes
			3.4.1 Examples of Application
		3.5 Rotating Disk Reactor (RDR) for Biodegradation of Textile  Dyes
			3.5.1 Examples of Application
	4 Notes
	References
Chapter 8: Denaturing Gradient Gel Electrophoresis (DGGE) Analysis of the Fungi Involved in Biodegradation
	1 Introduction
	2 Materials
		2.1 Fungal DNA Extraction
		2.2 For DGGE PCR
		2.3 Gel Casting/Loading/Running
		2.4 Staining and Visualization
	3 Methods
		3.1 DNA Extraction by the CTAB Method
		3.2 DGGE  PCR
		3.3 Gel Casting/Loading/Running
		3.4 Staining and Visualization
	4 Notes
	References
Chapter 9: Optimization of Mycoremediation Process for the Isolated Fungi
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation Media and Stock Solutions for the Mycoremediation
		3.2 Mycoremediation of the Xenobiotics
		3.3 High-Performance Liquid Chromatography of the Extracted Sample
		3.4 Calculation of the Mycoremediation Kinetics
	4 Notes
	References
Chapter 10: Protocol for Assessing Mycoremediation of Acidic Radioactive Wastes
	1 Introduction
	2 Materials
		2.1 Media and Solutions
			2.1.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Solutions
			2.1.2 Solution for DNA Detection Using Agarose Gel Electrophoresis
	3 Methods
		3.1 Elemental Analysis of Solid and Solution
		3.2 Samples Collection and Preparation
		3.3 Isolation of Active Fungi in Acidic Radioactive Wastes Remediation
		3.4 Screening of Fungal Isolates for Radioactive Wastes Remediation
		3.5 Efficiency of Filamentous Aquatic Fungi to Uptake Radioactive Cs-137 and Co-60 from Exposing Wastes
		3.6 Efficiency of Fungi to Uptake Radioactive Cs-137 and CO-60 from Exposing Wastes
		3.7 Factors Affecting Radionuclide Uptake
		3.8 Effect of Pigments in Radionuclide Uptake
		3.9 Identification of the Most Active Fungal Isolate
		3.10 Transmission Electron Microscope for Characterization
		3.11 Phenotype Characterization
		3.12 Protein Analysis of Fungal Strains and/or Supernatants Free Cells Using SDS-PAGE
	4 Notes
	References
Chapter 11: Protocol for Screening Low-Density Polyethylene (LDPE)-Degrading Soil Fungi Isolated from Urban Waste Dumping Sites
	1 Introduction
	2 Materials
		2.1 Culture Media
		2.2 Test Material
		2.3 Other Chemicals
	3 Methods
		3.1 Pretreatment, Sterilization, and Disinfection of LDPE
			3.1.1 UV Pretreatment
			3.1.2 Thermal Pretreatment
			3.1.3 Presterilization of LDPE Films and Pellets
			3.1.4 Disinfection of Fungal-Treated LDPE Films and Pellets
		3.2 Sampling of Fungi
		3.3 Isolation of Fungi
			3.3.1 Spread Plate Method
			3.3.2 Single Spore Isolation
			3.3.3 Hyphal Tip Isolation
		3.4 Screening of LDPE-Degrading Fungi
			3.4.1 Screening of Fungi Using mPDB Medium
			3.4.2 Screening of Fungi Using BHB Medium
			3.4.3 Recovery of Pellets and Determinations of the Average Percentage of Weight Loss (WLP)
		3.5 Assessment of Biodegradation Potential of LDPE Films by Selected Fungi
			3.5.1 Incubation of LDPE Films on Solid Medium
			3.5.2 Incubation of LDPE Films in the Liquid Medium
		3.6 Quantitative Assessment of LDPE Biodegradation
			3.6.1 Calculation of Percentage of Weight Loss (WLP)
			3.6.2 Calculation of Carbonyl Index (CI)
			3.6.3 Calculation of Percentage of Crystallinity
			3.6.4 Calculation of Relative Elongation and Relative Tensile Strength
		3.7 Qualitative Analysis of LDPE Biodegradation: Scanning Electron Microscopy (SEM)
	4 Notes
	References
Chapter 12: Production of Laccases from Agricultural Wastes: Strain Isolation and Selection, Enzymatic Profiling, and Lab-Scal...
	1 Introduction
		1.1 Occurrence of Laccase in Fungal Systems
	2 Materials
		2.1 Culture Media
	3 Methods
		3.1 Isolation of Promising Fungal Strains
		3.2 Qualitative Assays for Laccase Production
		3.3 Laccase Production in Solid-State Fermentation (SSF)
		3.4 Quantitative Laccase Activity and Protein Content Determination
		3.5 Conditioning of Enzyme Extracts: Partial Laccase Purification
			3.5.1 Aqueous Two-Phase Extraction (ATPE)
			3.5.2 Foam Fractionation (FF)
			3.5.3 Cross-Linking of Enzyme Aggregates (CLEAs)
		3.6 Concentration
		3.7 Affinity Chromatography: Final Laccase Purification
		3.8 Electrophoresis
	4 Notes
	References
Chapter 13: Bioremediation of Sugarcane Vinasse by Fungi-Based Biological Methods
	1 Introduction
	2 Materials
		2.1 Sugarcane Vinasse
		2.2 Fungi
		2.3 Fungi Cultivation
		2.4 Toxicity Tests
	3 Methods
		3.1 Sampling and Characterization of Sugarcane Vinasse
		3.2 Preparation of Fungi Spores
		3.3 Selection of Fungi Culture Conditions
		3.4 Vinasse Treatment by Fungi-Based Biological Methods
		3.5 Toxicity Assessed of Raw and Treated Vinasse Using Vegetable Cells (Wheat Seeds) as the Indicator Organism
		3.6 Toxicity Assessed of Raw and Treated Vinasse Using Mammalian Cells (Caco-2 Cell Line) as the Indicator Organism
	4 Notes
	5 Conclusion
	References
Chapter 14: Two-Dimensional Gel Electrophoresis: Discovering Isoenzymes for Mycoremediation
	1 Introduction
	2 Materials
		2.1 Fungal Growth and Supernatant Obtention
		2.2 Sample Preparation and Two-Dimensional Gel Electrophoresis
		2.3 Staining Step
	3 Methods
		3.1 Microbial Growth and Protein Extraction
		3.2 Preparation of Molecular Weight Marker
		3.3 Second-Dimensional Gel Electrophoresis on SDS-PAGE
		3.4 Staining Step
		3.5 Measurement of Protein Sizes
	4 Notes
	References
Chapter 15: Measurement of Ligninolytic Enzymes of Soil Treated with Bioaugmentation
	1 Introduction
	2 Materials
		2.1 Fungal Maintenance
		2.2 Bioaugmentation
		2.3 Enzymatic Extraction from Bioaugmented Soil
		2.4 Oxidative Activity Determination
		2.5 Hydrolytic Activity Determination
	3 Methods
		3.1 Bioaugmentation
		3.2 Enzymatic Extraction
		3.3 Oxidative Activity Determination
		3.4 Hydrolytic Activity Determination
	4 Notes
	References
Chapter 16: Whole Shotgun Proteomics and Its Role in Mycoremediation
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 Protein Quantification
		2.3 HPLC-MS/MS Analysis
	3 Methods
		3.1 Sample Preparation
		3.2 Protein Quantification
		3.3 Mass Spectrometry and Analysis
	4 Notes
	References
Chapter 17: Isolation and Selection of Tolerant Fungal Strains from Soil Polluted with Heavy Metals
	1 Introduction
	2 Materials
		2.1 Soil Sample Collection
		2.2 Determination of Total Chromium in Contaminated Soil
		2.3 Isolation of Chromium-Tolerant Fungi
			2.3.1 Preparation of Antibiotic Supplemented Isolation Medium: Modified Lee´s Minimal Medium
			2.3.2 Fungal Isolation from Soil
		2.4 Morphological Identification of Isolated Fungal Strains
		2.5 Fungal Tolerance Analysis
			2.5.1 Plate Screening Technique to Measure Fungi Tolerance
	3 Methods
		3.1 Soil Sample Collection
		3.2 Determination of Total Chromium in Polluted Soil
		3.3 Isolation of Chromium-Tolerant Fungi
			3.3.1 Preparation of Antibiotic Supplemented Isolation Medium: Modified Lee´s Minimal Medium
			3.3.2 Fungal Isolation from Soil
		3.4 Morphological Identification of Isolated Fungal Strains
		3.5 Fungal Tolerance Analysis
			3.5.1 Plate Screening Technique to Measure Fungi Tolerance
			3.5.2 Fungal Tolerance Determination
	4 Notes
	References
Chapter 18: Mycoremediation of Wastewater by Fungal Lipases
	1 Introduction
	2 Materials
		2.1 Obtaining Supernatant Rich in Lipase Enzyme
		2.2 Application of Lipase-Rich Supernatant in Water Contaminated with Fats and  Oils
	3 Methods
		3.1 Reactivation of Fungal Strain on  Agar
		3.2 Obtaining Supernatant Rich in Lipase Enzyme from Penicillium Fungus
		3.3 Effluent Treatment with Penicillium Lipase-Rich Supernatant
			3.3.1 Sampling and Preservation
			3.3.2 Mycoremediation Treatment
			3.3.3 Determination of Oils and Fats
	4 Notes
	References
Chapter 19: Bioaugmentation of Biomixtures with Consortia of Actinobacteria and Fungi for Improving Pesticides Removal
	1 Introduction
	2 Materials
		2.1 Microorganisms
		2.2 Culture Media
		2.3 Components of the Biomixture
		2.4 Pesticide
	3 Methods
		3.1 Evaluation of Antagonism Among Actinobacteria Strains and Fungi
		3.2 Inoculum Preparation
		3.3 Biomixtures Preparation
		3.4 Microbial Counts
		3.5 Lindane Analysis
		3.6 Kinetic Parameters of Lindane Removal
	4 Notes
	References
Chapter 20: Predictive Mycology for the Screening of White-Rot Fungi
	1 Introduction
	2 Materials
		2.1 Maintenance of Fungi
		2.2 Tolerance to PCBs
		2.3 Ligninolytic Enzyme Assay
	3 Methods
		3.1 Tolerance to PCBs
		3.2 Ligninolytic Enzyme Assay
		3.3 Modelling and Statistical Analysis
	4 Notes
	References
Chapter 21: Enzyme Biosensors for the Detection of Environmental Contaminants
	1 Introduction
	2 Materials
		2.1 Obtaining the Laccase Enzyme
		2.2 Conformation of the Enzymatic Biosensor
	3 Methods
		3.1 Reactivation of the Fungus on Agar
		3.2 Obtaining the Fungus Supernatant
		3.3 Preparation of the Enzyme To Be Used in the Biosensor
		3.4 Preparation and Loading of Screen-Printed Electrodes for Measurement
		3.5 Electrochemical Measurements
	4 Notes
	References
Index