Research on Nitrification and Related Processes, Part 2

Series Editors
......Page 1
Copyright
......Page 2
Contributors
......Page 3
Preface
......Page 9
Methods in Enzymology
......Page 11
Strategies to Determine Diversity, Growth, and Activity of Ammonia-Oxidizing Archaea in Soil......Page 40
Introduction......Page 41
Soil sampling and experimental design......Page 43
Nucleic acid extraction......Page 44
Reverse transcription and cDNA production......Page 46
PCR of 16S rRNA genes......Page 47
PCR amplification of amoA genes......Page 50
Analysis of acetyl-CoA carboxylase genes......Page 51
Community Composition and Diversity......Page 52
Nucleic acid fingerprinting......Page 54
Analysis of clone libraries......Page 55
High-throughput sequencing methods......Page 57
Changes in relative abundance......Page 58
Quantification of gene abundance......Page 59
Activity......Page 60
Estimating activity from gene abundance data......Page 62
Stable isotope probing......Page 63
Conclusions......Page 66
References......Page 68
Responses of Aerobic and Anaerobic Ammonia/Ammonium-Oxidizing Microorganisms to Anthropogenic Pollution in Coastal......Page 72
Introduction......Page 73
Selection of Sampling Sites and Physicochemical Characterization......Page 75
Genetic markers......Page 76
Extraction methods of total genomic DNA and clone library constructions......Page 79
The abundance of aerobic and anaerobic ammonia/ammonium-oxidizing microbes as determined by real-time fluorescent PCR (qP......Page 80
The diversity and richness of microbial community......Page 81
Phylogenetic lineages......Page 83
Preparation of the input files......Page 84
Explanation of UniFrac statistical results......Page 85
Relationship Between the Community Structure and Physicochemical Parameters......Page 87
Preparation of the input files......Page 88
Explanation of CCA statistical results......Page 89
Relationships Between the Community Change and the Environments......Page 91
Conclusions......Page 92
References......Page 93
Molecular and Stable Isotope Methods to Detect and Measure Anaerobic Ammonium Oxidation (Anammox) in Aquatic Ecosy......Page 100
Introduction......Page 101
Molecular Methods to Detect and Quantify Anammox Bacteria in Environmental Samples......Page 103
PCR protocols of 16S rRNA gene detection......Page 105
PCR protocol of hzo gene detection......Page 108
Quantitative PCR of anammox bacteria......Page 111
Stable Isotope Methods to Measure Anammox Rates in Environmental Samples......Page 114
Sediment slurries and whole water incubations......Page 115
Working toward in situ rates-Whole cores and ambient O2......Page 119
References......Page 121
Nitrogen Mineralization and Assimilation at Millimeter Scales......Page 127
Microbial Habitats in Soil......Page 128
Plant detritus......Page 129
Methodological Approaches......Page 130
General isotope pool dilution principles and procedures......Page 131
Physical separation followed by labeling......Page 132
Labeling followed by physical dissection......Page 134
Applications of SIMS analysis to soil microhabitats......Page 137
Combining N assimilation with microbial identification......Page 141
Sample preparation for SIMS......Page 142
NanoSIMS instrument use......Page 143
References......Page 145
Measurement of Carbon Dioxide, Methane, Nitrous oxide, and Water Potential in Soil Ecosystems......Page 151
Introduction......Page 152
Soil Gas Probes......Page 158
Data Analysis......Page 161
Soil Profile Analysis......Page 163
Recirculating versus static chambers......Page 164
Correlations Between Profile Concentrations and Surface Flux in Field Measurements......Page 165
Common Issues During FTIR Measurement......Page 168
Conclusions......Page 170
References......Page 171
Source Determination of Nitrous Oxide Based on Nitrogen and Oxygen Isotope Tracing......Page 174
Introduction......Page 175
Experimental setup......Page 178
Soil mineral N analyses......Page 179
Main assumptions......Page 180
Quantifying O exchange: The ERR approach......Page 181
Distinguishing N2O production pathways: 15N data analyses......Page 182
Distinguishing N2O production pathways: 18O data analyses......Page 183
Application of the ERR Principle in Nitrate Source Determination......Page 185
Discussion, Applications, and Future Directions......Page 187
References......Page 191
A Polyphasic Approach to Study Ecophysiology of Complex Multispecies Nitrifying Biofilms......Page 196
Introduction......Page 197
Microsensors......Page 198
Amperometric microsensors......Page 199
Potentiometric microsensors......Page 200
Estimation of Microbial Activities......Page 202
Limitations of Microsensor Measurements......Page 204
Methodology of MAR
-FISH......Page 205
Sample incubation with radioactive compound......Page 206
Sample fixation, washing, and preparation of slides......Page 207
Fish
......Page 208
Microscopic observation......Page 209
Application of Microsensors and MAR-FISH to Nitrifying Biofilms......Page 210
Ecophysiological Interaction Among Community Members......Page 213
References......Page 215
In Situ Techniques and Digital Image Analysis Methods for Quantifying Spatial Localization Patterns of Nitrifiers......Page 218
Introduction......Page 219
The Linear Dipole Algorithm......Page 223
The ``Inflate Algorithm´´......Page 230
In situ detection of nitrifiers by FISH......Page 234
Digital image analysis......Page 239
An Application Example: Spatial Analysis of Three Nitrifying Biofilm Populations......Page 241
References......Page 244
Investigating Nitrosomonas europaea Stress Biomarkers in Batch, Continuous Culture, and Biofilm Reactors......Page 249
Introduction......Page 251
Preventing O2 and NH3 limitations......Page 254
Experimental protocol and physiological measurements......Page 255
Transcriptional assays and sentinel gene expression......Page 259
Batch growth assays......Page 265
Fill and draw reactors......Page 266
Continuous growth reactors......Page 269
Identifying Stress Responses in Biofilms......Page 271
Conclusions......Page 274
References......Page 276
Nitrification of Raw or Used Water Using Expanded Bed Biofilm Reactor Technology......Page 279
Treatment of raw or used water......Page 280
EBBR technology......Page 282
Design and Operation of EBBRs......Page 284
Bioreactor design......Page 285
Biofilm thickness control......Page 287
pH control......Page 288
Bioreactor startup......Page 289
Miniature bioreactor for bioparticle rate measurement......Page 290
Measurement of Nitrification Performance......Page 291
Nitrite......Page 292
Measurement of dissolved oxygen (DO)......Page 293
Miniature EBBR......Page 294
Conclusions......Page 297
References......Page 298
Ammonia-Oxidizing Bacteria in Wastewater......Page 300
Introduction......Page 301
DNA Extraction......Page 302
Preparation of the qPCR standards......Page 303
PCR primers......Page 304
Fluorescence In Situ Hybridization......Page 306
Microscope slide......Page 307
Fluorescently labeled oligonucleotide probes......Page 308
FISH on slides......Page 310
Microscopy and AOB detection......Page 312
Conclusion......Page 314
References......Page 315
Genomics for Key Players in the N Cycle......Page 318
Introduction: The Genomic Guinea Pigs......Page 319
Sample and sequencer considerations......Page 323
Creating libraries for different platforms......Page 324
Sequencing in the ``Next-Gen´´ era......Page 326
Assembling sequence reads......Page 329
Genome closure and polishing......Page 333
Annotation......Page 335
Comparative analysis......Page 340
Outlook: The Next Frontier......Page 341
References......Page 342
Preparation of High-Molecular Weight DNA and Metagenomic Libraries from Soils and Hot Springs......Page 348
Introduction......Page 349
Terrestrial hot springs as habitat......Page 351
Construction of a metagenomic fosmid library: An overview......Page 352
Sampling and preservation of samples......Page 353
Extraction of cells from acidic hot spring mud samples......Page 354
Isolation of cells from soil samples......Page 355
Isolation of hmw DNA-Procedure 1......Page 356
Isolation of hmw DNA-Procedure 2......Page 357
Initial size analysis of the isolated DNA......Page 359
Purification of hmw DNA using a two-phased agarose gel in PFGE......Page 360
Isolation of hmw DNA from agarose matrix......Page 361
Isolation of hmw DNA from agarose matrix using agarase......Page 362
Isolation of hmw DNA from agarose matrix via electroelution......Page 363
Ligation of insert DNA to fosmid vector......Page 364
Infection of the E. coli host cells with lambda bacteriophages......Page 365
Quality testing of fosmid clones......Page 366
Growing, Picking, Replicating, and Storage of the Fosmid Library......Page 367
Screening of specific genes in the fosmid library......Page 369
General conclusions......Page 370
References......Page 371
Characterizing Bacterial Gene Expression in Nitrogen Cycle Metabolism with RT-qPCR......Page 374
Why Study Bacterial Transformation of Reactive Nitrogen in the Environment?......Page 375
RNA analyses have distinct advantages in determining microbial contributions to the nitrogen cycle......Page 376
Quality RNA is key......Page 377
PCR primers must be chosen (and chosen again) carefully......Page 380
Determining relative mRNA expression levels requires a reliable internal standard......Page 381
Protocol: Bacterial RNA isolation......Page 383
N-cycle organisms and reactive nitrogen transformation pathways......Page 385
Laboratory culture of aerobic N-cycle bacteria with emphasis on expression studies......Page 389
Using genome-informed metabolic reconstruction of catabolic pathways to select target genes......Page 390
Future Directions of the Approach......Page 393
Acknowledgments......Page 394
References......Page 395
The Utility of Functional Gene Arrays for Assessing Community Composition, Relative Abundance, and Distribution o......Page 402
Introduction......Page 403
Probe Selection......Page 404
Target Preparation......Page 408
Array Printing, Hybridization, and Scanning......Page 409
Factors That Influence Hybridization Results......Page 410
Array Applications......Page 413
Possibilities and Limitations......Page 418
Detailed Protocol for Functional Gene Microarrays Using Oligonucleotide Probes......Page 419
References......Page 423
Structure and Function of Formate-Dependent Cytochrome c Nitrite Reductase, NrfA......Page 426
Introduction......Page 427
Isolation of NrfA......Page 428
Crystallization of NrfA and the NrfHA Complex......Page 429
Structure Solution by Multiple-Wavelength Anomalous Dispersion......Page 431
Structure of NrfA......Page 432
Heme Group Arrangement......Page 435
Active Site Architecture......Page 437
Substrate Complexes, Activity Assays, and Reactivity......Page 438
Reaction Mechanism......Page 440
Electron Transfer Systems......Page 442
References......Page 445
Introduction......Page 450
Identification and classification of MCOs......Page 453
Detection of BCO in cell lysates......Page 454
Isolation......Page 456
Crystallization and structure solution......Page 457
References......Page 458
Assessing Variability in Gel-Based Proteomic Analysis of Nitrosomonas europaea......Page 461
Proteomics technologies......Page 462
Biological and technical variability......Page 465
Bacterial strain and culture conditions......Page 466
Second dimension separation: SDS-PAGE of IPG strips......Page 468
Image analysis......Page 469
Protein identification......Page 470
Chemostat stability and growth data......Page 471
Within-sample variability......Page 472
Between-sample variability......Page 474
Protein identification......Page 479
Recommendations for dealing with variability in proteomic studies......Page 480
References......Page 486
Nitrogen Metabolism and Kinetics of Ammonia-Oxidizing Archaea......Page 490
Introduction......Page 491
Culture media and growth conditions......Page 493
Nutrient measurements, cell counts, and protein quantification......Page 494
Microrespirometry Setup......Page 495
Stoichiometry and Kinetics of Ammonia Oxidation of N. maritimus and AOB......Page 499
Variability of Kinetic Constants in AOB and AOA......Page 505
Summary and Conclusions......Page 507
References......Page 508
Author Index
......Page 513
Subject Index
......Page 539