Experimental Methods in Wastewater Treatment

Mark C. M. van Loosdrecht; Per Halkjaer Nielsen; C. M. Lopez-Vazquez; Damir Brdjanovic

BOOK

Experimental Methods in Wastewater Treatment

Mark C. M. van Loosdrecht | Per Halkjaer Nielsen | C. M. Lopez-Vazquez | Damir Brdjanovic

(2016)

Additional Information

Book Details

ISBN: 978-1-78040-475-2
Edition: 1
Language: English
Pages: 362
Subjects: The environment
Water supply & treatment
Mining technology & engineering

Abstract

Over the past twenty years, the knowledge and understanding of wastewater treatment has advanced extensively and moved away from empirically based approaches to a fundamentally-based first principles approach embracing chemistry, microbiology, and physical and bioprocess engineering, often involving experimental laboratory work and techniques. Many of these experimental methods and techniques have matured to the degree that they have been accepted as reliable tools in wastewater treatment research and practice. For sector professionals, especially a new generation of young scientists and engineers entering the wastewater treatment profession, the quantity, complexity and diversity of these new developments can be overwhelming, particularly in developing countries where access to advanced level laboratory courses in wastewater treatment is not readily available. In addition, information on innovative experimental methods is scattered across scientific literature and only partially available in the form of textbooks or guidelines. This book seeks to address these deficiencies. It assembles and integrates the innovative experimental methods developed by research groups and practitioners around the world. Experimental Methods in Wastewater Treatment forms part of the internet-based curriculum in wastewater treatment at UNESCO-IHE and, as such, may also be used together with video records of experimental methods performed and narrated by the authors including guidelines on what to do and what not to do. The book is written for undergraduate and postgraduate students, researchers, laboratory staff, plant operators, consultants, and other sector professionals.

Section Title	Page	Action	Price
Cover	Cover
Table of contents	x
1. INTRODUCTION	1
References	5
2. ACTIVATED SLUDGE ACTIVITY TESTS	7
2.1 INTRODUCTION	7
2.2 ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL	9
2.2.1 Process description	9
2.2.2 Experimental setup	11
2.2.2.1 Reactors	11
Anaerobic conditions	12
Anoxic conditions	12
Aerobic conditions	13
Mixing	13
Temperature control	13
pH control	14
Sampling and dosing ports	15
2.2.2.2 Activated sludge sample collection	16
2.2.2.3 Activated sludge sample preparation	16
2.2.2.4 Substrate	17
2.2.2.5 Analytical procedures	19
PHA	19
Glycogen	21
2.2.2.6 Parameters of interest	22
2.2.3 EBPR batch activity tests: preparation	22
2.2.3.1 Apparatus	22
2.2.3.2 Materials	24
2.2.3.3 Media preparation	24
Real wastewater	24
Synthetic influent media or substrate	24
Nitrate or nitrite solution	25
Washing media	25
Formaldehyde solution	25
ATU (Allyl-N-thiourea) solution	25
Acid and base solutions	25
2.2.3.4 Material preparation	25
2.2.3.5 Activated sludge preparation	28
2.2.4 Batch activity tests: execution	29
2.2.4.1 Anaerobic EBPR batch activity tests	30
Test EBPR.ANA.1 Anaerobic batch EBPR tests performed under the absence of an electron donor	31
Test EBPR.ANA.2 Anaerobic batch EBPR tests performed under a defined addition of an electron donor	31
Test EBPR.ANA.3 Anaerobic batch EBPR tests performed after the addition of an electron donor in excess	32
2.2.4.2 Anoxic EBPR batch tests	33
Test EBPR.ANOX.1 Single anoxic EBPR batch tests	33
Test EBPR.ANOX.2 Combined anaerobic-anoxic EBPR batch tests	34
2.2.4.3 Aerobic EBPR batch tests	34
Test EBPR.AER.1 Single aerobic EBPR test	35
Test EBPR.AER.3 Combined anaerobic-anoxic-aerobic EBPR batch tests in series	36
Test EBPR.AER.4 Combined anaerobic-anoxic-aerobic EBPR batch tests in parallel	36
2.2.5 Data analysis	36
2.2.5.1 Estimation of stoichiometric parameters	36
2.2.5.2 Estimation of kinetic parameters	41
2.2.6 Data discussion and interpretation	42
2.2.6.1 Anaerobic batch activity tests	42
2.2.6.2 Aerobic batch activity tests	45
2.2.6.3 Anoxic batch activity tests	46
2.2.7 Example	47
2.2.7.1 Description	47
2.2.7.2 Data analysis	47
2.2.8 Additional considerations	51
2.2.8.1 GAO occurrence in EBPR systems	51
2.2.8.2 The effect of carbon source	51
2.2.8.3 The effect of temperature	51
2.2.8.4 The effect of pH	52
2.2.8.5 Denitrification by EBPR cultures	52
2.2.8.6 Excess/shortage of intracellular compounds	52
2.2.8.7 Excessive aeration	53
2.2.8.8 Shortage of essential ions	53
2.2.8.9 Toxicity/inhibition	53
2.3 BIOLOGICAL SULPHATE-REDUCTION	54
2.3.1 Process description	54
2.3.2 Sulphide speciation	56
2.3.3 Effects of environmental and operating conditions on SRB	57
2.3.3.1 Carbon source	57
2.3.3.2 COD to SO42- ratio	58
2.3.3.3 Temperature	58
2.3.3.4 pH	59
2.3.3.5 Oxygen	59
2.3.4 Experimental setup	60
2.3.4.1 Estimation of volumetric and specific rates	60
2.3.4.2 The reactor	60
2.3.4.3 Mixing	61
2.3.4.4 pH control	61
2.3.4.5 Temperature control	61
2.3.4.6 Sampling and dosing ports	62
2.3.4.7 Sample collection	62
2.3.4.8 Media	62
2.3.5 Analytical procedures	63
2.3.5.1 CODorganics and CODtotal	63
2.3.5.2 Sulphate	64
2.3.5.3 Sulphide	64
2.3.6 SRB batch activity tests: preparation	65
2.3.6.1 Apparatus	65
2.3.6.2 Materials	65
2.3.6.3 Media	65
Real wastewater	65
Synthetic media or substrate	65
2.3.6.4 Material preparation	66
2.3.6.5 Mixed liquor preparation	67
2.3.6.6 Sample collection and treatment	68
Filtered sample	68
Filtered sample with NaOH	68
Gas sample	68
Biomass sample	68
2.3.7 Batch activity tests: execution	68
Test SRB.ANA.1 Anaerobic SRB activity test	69
Test SRB.ANA.2 Anaerobic SRB activity test	69
2.3.8 Data analysis	69
2.3.8.1 Mass balances and calculations	69
2.3.8.2 Data discussion and interpretation	70
2.3.9 Example	70
2.3.10 Practical recommendations	72
2.4 BIOLOGICAL NITROGEN REMOVAL	73
2.4.1 Process description	73
2.4.1.1 Nitrification	74
2.4.1.2 Denitrification	75
2.4.1.3 Anaerobic ammonium oxidation (anammox)	76
2.4.2 Process-tracking alternatives	76
2.4.2.1 Chemical tracking	77
2.4.2.2 Titrimetric tracking	77
2.4.2.3 Manometric tracking	78
2.4.3 Experimental setup	79
2.4.3.1 Reactors	79
2.4.3.2 Instrumentation for titrimetric tests	79
2.4.3.3 Instrumentation for manometric tests	80
2.4.3.4 Activated sludge sample collection	81
2.4.3.5 Activated sludge sample preparation	82
2.4.3.6 Substrate	82
2.4.3.7 Analytical procedures	83
2.4.3.8 Parameters of interest	83
Nitrification	83
Denitrification	84
Anammox	85
2.4.3.9 Type of batch tests	86
2.4.4 Nitrification batch activity tests: preparation	86
2.4.4.1 Apparatus	86
2.4.4.2 Materials	86
2.4.4.3 Media preparation	86
Real wastewater	86
Titration solutions	86
Ammonium and nitrite stock solutions	87
Allyl-N-thiourea (ATU)	87
Acid and base solutions	87
Synthetic medium	87
Washing media	87
2.4.5 Nitrification batch activity tests: execution	87
Test NIT.CHE Nitrification chemical test: assessing the maximum ammonium oxidation rate	87
Activated sludge preparation	87
Execution of the test	88
Data analysis	88
Test NIT.TIT.1 Nitrification titration test: assessing the maximum ammonium oxidation rate	89
Activated sludge preparation	89
Execution of the test	89
Data analysis	89
Test NIT.TIT.2 Nitrification titration test: assessing the maximum ammonium and nitrite oxidation rates	90
Activated sludge preparation	90
Execution of the test	90
Data analysis	90
2.4.6 Denitrification batch activity tests: preparation	92
2.4.6.1 Apparatus	92
2.4.6.2 Materials	93
2.4.6.3 Working solutions	93
Real wastewater	93
Carbon source solution	93
Nitrate or nitrite solutions	93
Washing media	93
Acid and base solutions	93
Nutrient solution	93
2.4.6.4 Material preparation	93
2.4.7 Denitrification batch activity tests: execution	93
Test DEN.CHE.1 Denitrification chemical test: assessing the maximum denitrification rate and the anoxic growth yield in the presence of a specific carbon source	93
Activated sludge preparation	93
Execution of the test	94
Data analysis	94
Test DEN.CHE.2 Denitrification chemical test: assessing the denitrification potential of wastewater	95
Activated sludge preparation	95
Test execution	95
Data analysis	95
Test DEN.MAN Denitrification manometric test: assessing the denitrification kinetic rate	96
Activated sludge preparation	96
3. RESPIROMETRY	133
3.1 INTRODUCTION	133
3.1.1 Basics of respiration	134
3.1.2 Basics of respirometry	135
3.2 GENERAL METHODOLOGY OF RESPIROMETRY	136
3.2.1 Basics of respirometric methodology	136
3.2.2 Generalized principles: beyond oxygen	136
3.2.2.1 Principles based on measuring in the liquid phase	136
Static gas, static liquid (LSS)	137
Flowing gas, static liquid (LFS)	137
Static gas, flowing liquid (LSF)	138
Flowing gas, flowing liquid (LFF)	138
3.2.2.2 Principles based on measuring in the gas phase	138
Static gas, static liquid (GSS)	139
Flowing gas, static liquid (GFS)	140
Static gas, flowing liquid (GSF)	140
Flowing gas, flowing liquid (GFF)	140
3.3 EQUIPMENT	141
3.3.1 Equipment for anaerobic respirometry	141
3.3.1.1 Biogas composition	141
Measuring the biogas composition and correcting the measured flow	141
Removing other gases from the biogas	142
3.3.1.2 Measuring the gas flow	142
Manometric methods	142
Volumetric methods	142
3.3.2 Equipment for aerobic and anoxic respirometry	143
3.3.2.1 Reactor	143
3.3.2.2 Measuring arrangement	143
3.3.2.3 Practical implementation	144
Liquid phase, static gas, static liquid (LSS) principle	144
3.4 WASTEWATER CHARACTERIZATION	150
3.4.1 Biomethane potential (BMP)	150
3.4.1.1 Purpose	150
3.4.1.2 General	150
3.4.1.3 Test execution	151
3.4.1.4 Data processing	151
3.4.1.5 Recommendations	151
Pressure and temperature correction	151
Methane diffusion	152
pH indicator dye for the scrubbing solution	152
Inoculum activity	152
Micro and macro nutrients	152
Oxygen inhibition	152
Gas tightness	152
Alkaline scrubbers	152
3.4.2 Biochemical oxygen demand (BOD)	152
3.4.2.1 Purpose	152
3.4.2.2 General	152
3.4.2.3 Test execution	153
BOD test with a LSS respirometer	153
Dilution	153
Seeding	153
Blank	154
DO measurement	154
Data processing	154
Recommendations	155
A BOD test with a GFS respirometer	155
Recommendations	156
3.4.3 Short-term biochemical oxygen demand (BODst)	157
3.4.3.1 Test execution	158
3.4.3.2 Calculations	160
3.4.4 Toxicity and inhibition	160
3.4.4.1 Purpose	160
3.4.4.2 Test execution	160
3.4.4.3 Calculations	161
3.4.4.4 Biodegradable toxicants	162
3.4.5 Wastewater fractionation	163
3.4.5.1 Readily biodegradable substrate (SB)	166
3.4.5.2 Slowly biodegradable substrate (XCB)	167
3.4.5.3 Heterotrophic biomass (XOHO)	168
3.4.5.4 Autotrophic (nitrifying) biomass (XANO)	168
3.4.5.5 Ammonium (SNHx)	168
3.4.5.6 Organic nitrogen fractions (XCB,N and SB,N)	168
3.5 BIOMASS CHARACTERIZATION	169
3.5.1 Volatile suspended solids	169
3.5.2 Specific methanogenic activity (SMA)	169
3.5.2.1 Purpose	169
3.5.2.2 General	169
3.5.2.3 Test execution	169
3.5.2.4 Data processing	170
3.5.3 Specific aerobic and anoxic biomass activity	171
3.5.3.1 Maximum specific nitrification rate (AUR)	171
3.5.3.2 Maximum specific aerobic heterotrophic respiration rate (OUR)	173
3.5.3.3 Maximum specific denitrification rate (NUR)	173
References	175
4. OFF-GAS EMISSION TESTS	177
4.1 INTRODUCTION	177
4.2 SELECTING THE SAMPLING STRATEGY	178
4.2.1 Plant performance	178
4.2.2 Seasonal variations in emissions	178
4.2.3 Sampling objective	179
4.3 PLANT ASSESSMENT AND DATA COLLECTION	179
4.3.1. Preparation of a sampling campaign	179
4.3.2 Sample identification and data sheet	180
4.3.3 Factors that can limit the validity of the results	181
4.3.4 Practical advice for analytical measurements	181
4.3.5 General methodology for sampling	182
4.3.6 Sampling in the framework of the off-gas measurements	183
4.3.7. Testing and measurements protocol	185
4.4 EMISSION MEASUREMENTS	185
4.5 N2O MEASUREMENT IN OPEN TANKS	186
4.5.1 Protocol for measuring the surface flux of N2O	188
4.5.1.1 Equipment, materials and supplies	188
4.5.1.2 Experimental procedure	188
4.5.1.3 Sampling methods for nitrogen GHG emissions	189
The gas-phase sampling method in aerobic zones	189
Determination of the gas flow rate from the flux chamber in aerobic zones	190
The gas-phase sampling method in anoxic zones	190
Determination of the gas flow rate from the flux chamber in the anoxic zone	190
Continuous and real-time gas measurement	190
Principles of real-time N2O and CH4 measurements	191
4.5.1.4 Direct measurement of the liquid-phase N2O content	191
4.6 MEASUREMENT OF OFF-GAS FLOW IN OPEN TANKS	191
4.6.1 Protocol for aerated or aerobic zone	192
4.6.2 Protocol for non-aerated zones	192
4.7 AQUEOUS N2O and CH4 CONCENTRATION DETERMINATION	192
4.7.1 Measurement protocol for dissolved N2O measurement using polarographic electrodes	193
4.7.1.1 Equipment	193
4.7.1.2 Experimental procedure	193
4.7.2 Measurement protocol for dissolved gasses using gas chromatography	194
4.7.3 Measurement protocol for dissolved gas measurement by the salting-out method	194
4.7.3.1 Equipment	195
4.7.3.2 Sampling procedure	195
4.7.3.3 Measurement procedure	195
Measurement of the volume expansion due to pressure build-up in the bottle	195
Measurement of the methane or the nitrous oxide concentration in the headspace	195
Measurement of the headspace of the serum bottle	195
4.7.3.4 Calculations	196
Volume	196
Amount of methane	196
Concentration	196
4.7.4 Measurement protocol for dissolved gas measurement by the stripping method	196
4.7.4.1 Operational principle	196
4.7.4.2 Equipment	197
4.7.4.3 Calibration batch test	198
4.7.4.4 Measurement accuracy	198
4.7.4.5 Calculation of the N2O formation rate in the stripping device	198
4.8 DATA ANALYSIS AND PROCESSING	199
4.8.1 Determination of fluxes	199
4.8.2 Determination of aggregated emission fractions	199
4.8.3 Calculation of the emission factors	200
References	200
5. DATA HANDLING AND PARAMETER ESTIMATION	201
5.1 INTRODUCTION	201
5.2 THEORY AND METHODS	202
5.2.1 Data handling and validation	202
5.2.1.1 Systematic data analysis for biological processes	202
5.2.1.2 Degree of reduction analysis	203
5.2.1.3 Consistency check of the experimental data	204
5.2.2 Parameter estimation	205
5.2.2.1 The manual trial and error method	205
5.2.2.2 Formal statistical methods	205
The least squares method	206
The covariance matrix of parameter estimators	206
5.2.3 Uncertainty analysis	209
5.2.3.1 Linear error propagation	209
5.2.3.2 The Monte Carlo method	209
5.2.4 Local sensitivity analysis and identifiability analysis	210
5.2.4.1 Local sensitivity analysis	210
5.2.4.2 Identifiability analysis using the collinearity index	210
5.3 METHODOLOGY AND WORKFLOW	211
5.3.1 Data consistency check using an elemental balance and a degree of reduction analysis	211
5.3.2 Parameter estimation workflow for the non-linear least squares method	212
5.3.3 Parameter estimation workflow for the bootstrap method	212
5.3.4 Local sensitivity and identifiability analysis workflow	213
5.3.5 Uncertainty analysis using the Monte Carlo method and linear error propagation	213
5.4 ADDITIONAL EXAMPLES	214
5.5 ADDITIONAL CONSIDERATIONS	232
Best practice in parameter estimation	232
Best practice in uncertainty analysis	233
References	233
6. SETTLING TESTS	235
6.1 INTRODUCTION	235
6.2 MEASURING SLUDGE SETTLEABILITY IN SSTs	236
6.2.1 Sludge settleability parameters	237
6.2.1.1 Goal and application	237
6.2.1.2 Equipment	237
6.2.1.3 The Sludge Volume Index (SVI)	237
6.2.1.4 The Diluted Sludge Volume Index (DSVI)	237
6.2.1.5 The Stirred Specific Volume Index (SSVI3.5)	238
6.2.2 The batch settling curve and hindered settling velocity	238
6.2.2.1 Goal and application	238
6.2.2.2 Equipment	239
6.2.2.3 Experimental procedure	239
6.2.2.4 Interpreting a batch settling curve	240
6.2.2.5 Measuring the hindered settling velocity	241
6.2.3 vhs-X relation	241
6.2.3.1 Goal and application	241
6.2.3.2 Equipment	242
6.2.3.3 Experimental procedure	242
6.2.3.4 Determination of the zone settling parameters	243
6.2.3.5 Calibration by empirical relations based on SSPs	244
6.2.4 Recommendations for performing batch settling tests	245
6.2.4.1 Shape and size of the batch reservoir	245
6.2.4.2 Sample handling and transport	245
6.2.4.3 Concentration range	245
6.2.4.4 Measurement frequency	245
6.2.5 Recent advances in batch settling tests	245
6.3 MEASURING FLOCCULATION STATE OF ACTIVATED SLUDGE	246
6.3.1 DSS/FSS test	246
6.3.1.1 Goal and application	246
6.3.1.2 Equipment	246
General	246
ESS test	246
DSS test	246
FSS test	246
6.3.1.3 DSS test	246
6.3.1.4 FSS test	247
6.3.1.5 Interpretation of a DSS/FSS test	248
High DSSi low FSS	249
High DSSi high FSS	249
Low DSSi low FSS	249
Low DSSi high FSS	249
6.3.2 Recommendations	249
6.3.2.1 Flocculation conditions	249
6.3.2.2 Temperature influence	249
6.3.2.3 Supernatant sampling	249
6.3.3 Advances in the measurement of the flocculation state	250
6.4 MEASURING THE SETTLING BEHAVIOUR OF GRANULAR SLUDGE	250
6.4.1 Goal and application	250
6.4.2 Equipment	251
6.4.3 Density measurements	251
6.4.4 Granular biomass size determination	252
6.4.4.1 Sieving	252
6.4.4.2 Image analyser	253
6.4.5 Calculating the settling velocity of granules	253
6.4.6 Recommendations	254
6.4.6.1 Validation of results	254
6.4.6.2 Application for flocculent sludge	255
6.5 MEASURING SETTLING VELOCITY DISTRIBUTION IN PSTs	255
6.5.1 Introduction	255
6.5.2 General principle	255
6.5.3 Sampling and sample preservation	256
6.5.4 Equipment	256
6.5.5 Analytical protocol	257
6.5.6 Calculations and result presentation	258
6.5.6.1 Mass balance check	258
6.5.6.2 Calculation of the settling velocity distribution	258
6.5.6.3 Recommendations	259
References	260
7. MICROSCOPY	263
7.1 INTRODUCTION	263
7.2 THE LIGHT MICROSCOPE	263
7.2.1 Standard applications of light microscopy	265
7.2.2 Low power objective	265
7.2.3 High power objective	265
7.2.4 Immersion objective	265
7.2.5 Important considerations	266
7.2.6 Bright-field and dark-field illumination	266
7.2.7 Fluorescence microscopy	267
7.2.8 Confocal laser scanning microscopy	269
7.3 MORPHOLOGICAL INVESTIGATIONS	269
7.3.1 Microscopic identification of filamentous microorganisms	270
7.3.2 Identification of protozoa and metazoa	271
7.4 EXAMINING ACTIVATED SLUDGE SAMPLES MICROSCOPICALLY	272
7.4.1 Mounting the activated sludge sample	272
7.4.2 Gram staining	273
7.4.2.1 Reagents and solutions for Gram staining	273
Crystal violet solution	273
Gram’s iodine solution	274
Counterstain	274
Decolourizing solution	274
7.4.2.2 Procedure	274
7.4.3 Neisser staining	274
7.4.3.1 Reagents and solutions for Neisser staining	274
Methylene blue solution	274
Crystal violet solution	274
Counter-staining solution	274
Working solution	275
7.4.3.2 Procedure	275
7.4.4 DAPI staining	275
7.4.4.1 Reagents and solutions for DAPI staining	275
DAPI stock solution	275
7.4.4.2 Procedure	275
7.4.5 CTC staining	276
7.4.5.1 Reagents and solutions for CTC staining	276
7.4.5.2 Procedure	276
7.5 FLUORESCENCE in situ HYBRIDIZATION	276
7.5.1 Reagents and solutions for FISH	277
Fixative (8 % Paraformaldehyde, PFA) for Gram-negative cells	277
Lysozyme for cell permeabilisation	277
Proteinase K for cell permeabilisation	278
3 × phosphate-buffered saline (3 x PBS)	278
Tris-EDTA buffer (TE buffer)	278
1 M Tris-HCl, pH 8.0	278
5 M NaCl	278
Sterile distilled H2O (dH2O)	278
10 % Sodium dodecylsulfate (10 % SDS)	278
0.5 M EDTA	278
7.5.2 Procedure	278
7.6 COMBINED STAINING TECHNIQUES	280
7.6.1 FISH – DAPI staining	281
7.6.1.1 Reagents and solutions for DAPI staining	281
DAPI stock solution and storage	281
Reagents used for FISH	281
7.6.1.2 Procedure	281
7.6.2 FISH – PHA staining	282
7.6.2.1 Reagents and solutions for PHA staining	282
Reagents used for PHA staining	282
Reagents used for FISH	282
7.6.2.2 Procedure	282
References	282
8. MOLECULAR METHODS	285
8.1 INTRODUCTION	285
8.2 EXTRACTION OF DNA	286
8.2.1 General considerations	286
8.2.2 Sampling	286
8.2.3 DNA extraction	286
8.2.3.1 Cell lysis	286
8.2.3.2 Nuclease activity inhibition and protein removal	287
8.2.3.3 Purification	287
8.2.3.4 Elution and storage	287
8.2.4 Quantification and integrity	287
8.2.5 Optimised DNA extraction from wastewater activated sludge	288
8.2.5.1 Materials	288
8.2.5.2 DNA Extraction	288
Bead-beating	288
Protein precipitation and binding of DNA to matrix	288
DNA washing and elution	289
8.3 REAL-TIME QUANTITATIVE PCR (qPCR)	289
8.3.1 General considerations	289
8.3.2 Materials	291
Primers	291
Real-time thermal cycler	292
qPCR reagents	292
Equipment for measuring DNA concentration	292
8.3.3 Methods	292
Preparation of qPCR standards	292
Sample preparation	293
qPCR reaction setup	293
8.3.4 Data handling	294
Determination of sample copy numbers	294
Evaluation of PCR efficiency	294
8.3.5. Data output and interpretation	294
Extraction of nucleic acids is biased	294
Quality of the template DNA	294
Specificity of broad-range qPCR assays	295
Amplification of extracellular DNA (eDNA)	295
Variation in the gene copy number	295
8.3.6 Troubleshooting	295
The sample contains PCR inhibitors	295
The primer or probe design is not optimal	295
Inaccurate sample and reagent pipetting	295
8.3.7 Example	295
8.3.7.1 Samples	295
8.3.7.2 qPCR reaction setup	296
8.3.7.3 Results	296
8.4 AMPLICON SEQUENCING	297
8.4.1 General considerations	297
8.4.2 The 16S rRNA gene as a phylogenetic marker gene	297
8.4.3 PCR amplification	299
8.4.3.1 PCR reaction	299
8.4.3.2 PCR biases	300
8.4.3.3 Primer choice	300
8.4.4 DNA sequencing	301
8.4.4.1 Sequencing platform	301
8.4.4.2 Sequencing depth	301
8.4.5 Bioinformatic processing	301
8.4.5.1 Available software	301
8.4.5.2 Raw data	302
8.4.5.3 Quality scores and filtering	303
8.4.5.4 Merging paired-end reads	303
8.4.5.5 OTU clustering	303
8.4.5.6 Chimera detection and removal	304
8.4.5.7 Taxonomic classification	304
8.4.5.8 The OTU table	304
8.4.6 Data analysis	304
8.4.6.1 Defining the goal of the data analysis	304
8.4.6.2 Data validation and sanity check	305
8.4.6.3 Communities or individual species?	305
The community perspective	305
The species perspective	305
8.4.6.4 Identifying core and transient species	306
8.4.6.5 Explorative analysis using multivariate statistics.	306
8.4.6.6 Correlation analysis	307
8.4.6.7 Effect of treatments on individual species	307
8.4.7 General observations	307
8.4.7.1 A relative analysis	307
8.4.7.2 Copy number bias	307
8.4.7.3 Primer bias	307
8.4.7.4 Standardization	308
8.4.7.5 Impact of the method	308
8.4.8 Protocol: Illumina V1-3 16S rRNA amplicon libraries	308
8.4.8.1 Apparatus	308
8.4.8.2 Materials	308
8.4.8.3. Protocol	309
Sample DNA quality control and dilution (2.5 h)	309
Library PCR (2.0 h)	309
Library cleanup (2.0 h)	310
Library quality control (1.5 h)	311
Library pooling (2.0 h)	311
Storage and transport	311
8.4.9 Interpretation and troubleshooting	311
8.4.9.1 Sample DNA quality control and dilution	311
8.4.9.2 Library PCR	312
8.4.9.3 Library cleanup	312
8.4.9.4 Library quality control	313
8.4.9.5 Library pooling	314
8.4.9.6 Pool quality control and dilution	314
8.4.9.7 Storage	314
8.4.10 Protocol: Illumina V1-3 16S amplicon sequencing	314
8.4.10.1 Apparatus	314
8.4.10.2 Reagents	314
8.4.10.3 Protocol	314
Prepare MiSeq (2.0 h)	314
Prepare sequencing libraries (1.0 h)	315
Load the sample and primers on the reagent cartridge	315
8.4.10.4. Interpretation and troubleshooting	315
Prepare MiSeq and metadata	315
Prepare sequencing libraries	316
Load the sample and primers on the reagent cartridge	316
Sequencing	317
8.4.11 Design of Illumina 16S amplicon sequencing adaptors	317
8.5 OTHER METHODS	319
References	320
SYMBOLS AND ABBREVIATIONS	325

Experimental Methods in Wastewater Treatment

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