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Environmental
Biotechnology
About the Authors
Bruce E. Rittmann, Ph.D., is Regents' Professor of Environmental
Engineering and Director of the Biodesign Swette Center for
Environmental Biotechnology at Arizona State University. He is the
recipient of the Clarke Prize for Outstanding Achievement in Water
Science and Technology and the Stockholm Water Prize. Dr. Rittmann
is a Distinguished Member of the American Society of Civil Engineers
and a Member of the National Academy of Engineering.

Perry L. McCarty, Sc.D., is Silas H. Palmer Professor of Civil Engi-

neering Emeritus at Stanford University. He is the recipient of the
Clarke Prize for Outstanding Achievement in Water Science and
Technology, the Stockholm Water Prize, and the Tyler Environmental
Prize. Dr. McCarty is a Distinguished Member of the American
Society of Civil Engineers and a Member of the National Academy
of Engineering and of the American Academy of Arts and Sciences.

Cover Photo Credits

Bottom left image: Deer Island Waste Water Treatment Plant, used with
permission from the Massachusetts Water Resources Authority.
Bottom right image: Fluorescent in situ hybridization (FISH) photo-
micrograph of a partial-nitritation-anammox granule, provided by
Dr. Satoshi Okabe of Hokkaido University, Japan.
Environmental
Biotechnology
Principles and Applications

Bruce E. Rittmann, Ph.D.

Arizona State University
Tempe, Arizona

Perry L. McCarty, Sc.D.

Stanford University
Stanford, Californw

Second Edition

New York Chkago San Francisco

Athens London Madrid
Mexico City Milan New Delhi
Singapore Sydney Toronto
Copyright© 2020, 2001 by McGraw-Hill Education. All rights reserved Except as permitted under the United States Copyright
Act ofl 976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or
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ISBN: 978-1-26-044161-1
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 Contents
Preface....................................................... xv
1 Moving Toward Sustainability. . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . 1
1.1 Water Uses and Resources................................. 1
1.2 Wastewater's Resources. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Climate Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.4 Sustainability............................................ 4
1.5 The Role of Environmental Biotechnology. . . . . . . . . . . . . . . . . . . 5
1.6 Organization of the Book. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2 Basics of Microbiology. • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . 9
2.1 The Microbial Cell........................................ 10
2.2 Microbial Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3 Pro.karyotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.1 Bacterial and Archaeal Cell Structure and Function . . . . . 15
2.3.2 Phylogenic Lineages of Bacteria . . . . . . . . . . . . . . . . . . . . . . 25
2.3.3 Phylogenic Lineages of Archaea. . . . . . . . . . . . . . . . . . . . . . 28
2.4 Eukarya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.4.1 Fungi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.4.2 Algae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.4.3 Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4.4 Other Multicellular Microorganisms. . . . . . . . . . . . . . . . . . 41
2.5 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.6 Infectious Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3 Biochemistry, Metabolism, Genetics, and Information Flow. . . . . . . . 51
3.1 Biochemistry............................................ 51
3.1.1 Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.1.2 Enzyme Reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.1.3 Regulating Enzyme Activity. . . . . . . . . . . . . . . . . . . . . . . . . 59
3.2 Energy Capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.2.1 Electron and Energy Carriers . . . . . . . . . . . . . . . . . . . . . . . . 59
3.2.2 Energy and Electron Investments..................... 61
3.3 Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.3.1 Catabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.3.2 Anabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.3.3 Metabolism and Trophic Groups . . . . . . . . . . . . . . . . . . . . . 86
3.4 Genetics and Information Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3.4.1 Deoxyribonucleic Acid (DNA) . . . . . . . . . . . . . . . . . . . . . . . 89

y
Yi Contents

3.4.2 The Chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

3.4.3 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3.4.4 DNA Replication.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.4.5 Ribonucleic Acid (RNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.4.6 Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
3.4.7 Messenger RNA (mRNA) . . . . . . . . . . . . . . . . . . . . . . . . . . 96
3.4.8 Transfer RNA (tRNA).............................. 96
3.4.9 Translation and the Ribosomal RNA (rRNA) . . . . . . . . . . 97
3.4.10 Translation....................................... 98
3.4.11 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
3.4.12 Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
3.4.13 The Basics of Phylogenetic Classification............. 102
3.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.6 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
3.7 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4 Microbial Ecology.......................................... ... 109
4.1 Selection................................................ 110
4.2 Exchange of Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.2.1 Exchange of Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.2.2 Exchange of Genetic Information. . . . . . . . . . . . . . . . . . . . . 116
4.2.3 Growth Factors.................................... 117
4.2.4 Exchange of Chemical Signals . . . . . . . . . . . . . . . . . . . . . . . 117
4.3 Adaptation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4.4 Tools to Study Microbial Ecology. . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.4.1 Traditional Enrichment Tools . . . . . . . . . . . . . . . . . . . . . . . . 120
4.4.2 Molecular Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.4.3 Genomics Methods Based on the Ribosomal RNA...... 123
4.4.4 Genomics Methods Based on the Ribosomal DNA . . . . . . 126
4.4.5 Diversity Analysis of Genomics Results . . . . . . . . . . . . . . . 135
4.4.6 Functional Genomics Analysis . . . . . . . . . . . . . . . . . . . . . . . 136
4.4.7 Transcriptomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
4.4.8 Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
4.4.9 Functional Prediction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
4.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.6 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.7 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
5 Stoichiometry and Energetics................................... 143
5.1 An Example Stoichiometric Equation....................... 143
5.2 An Empirical Formula for Microbial Cells . . . . . . . . . . . . . . . . . . . 144
5.3 Formulations for Cells Containing Storage Products . . . . . . . . . . 148
5.4 Substrate Partitioning and Cellular Yield . . . . . . . . . . . . . . . . . . . . 148
5.5 Overall Reactions for Biological Growth. . . . . . . . . . . . . . . . . . . . . 150
5.6 Fermentation Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
5.6.1 Simple Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
5.6.2 Mixed Fermentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
 Ca nt ent s vii

5.7 Energetics of Bacterial Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

5.7.1 Free Energy of the Energy Reaction.... . . . . . . . . . . . . . . . 164
5.7.2 Microbial Yield Coefficient and Reaction Energetics..... 167
5.7.3 Oxidized Nitrogen Sources.......................... 173
5.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
5.9 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
6 Microbial Kinetics. . . . . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . 181
6.1 Basic Rate Expressions... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
6.2 Estimating Parameter Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
6.3 Basic Mass Balances................................ ...... 191
6.4 Mass Balances on Inert Biomass and Volatile Suspended Solids. 197
6.5 Microbial Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
6.6 Input of Active Biomass............................. ...... 202
6.7 Nutrients and Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . 204
6.8 CSTR Summary Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
6.9 Hydrolysis of Particulate and Polymeric Substrates. . . . . . . . . . . 207
6.10 Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
6.11 Additional Rate Expressions............................... 215
6.12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
6.13 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
7 Biofilm Kinetics . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . 223
7.1 Microbial Aggregation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
7.2 Why Do Biofilms Form?................................... 223
7.3 The Idealized Biofilm.................................. ... 224
7.3.1 Substrate Phenomena............................... 226
7.3.2 Illustration for First-Order Kinetics................... 227
7.3.3 General Solution When S,. Is Known. . . . . . . . . . . . . . . . . . 229
7.3.4 The Biofilm Mass Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
7.4 The Steady-State Biofilm.................................. 230
7.5 The Steady-State-Biofilm Solution . . . . . . . . . . . . . . . . . . . . . . . . . . 231
7.6 Estimating Parameter Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
7.7 Average Biofilm SRT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
7.8 Completely Mixed Biofilm Reactor . . . . . . . . . . . . . . . . . . . . . . . . . 240
7.9 Inert Biomass, Nutrients, and Electron Acceptor. . . . . . . . . . . . . . 243
7.10 Trends in CMBR Performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
7.11 Normalized Surface Loading....................... ....... 247
7.12 Nonsteady-State Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
7.13 Special-Case Biofilm Solutions............................. 259
7.13.1 Deep Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
7.13.2 Zero-Order Kinetics............................... 260
7.14 Numerical Modeling of Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
7.15 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
7.16 Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
8 Microbial Products . . . . . • • . . . . . . . • • . . . . . . • • . . . . . . . • • . . . . . . . • • . . 273
8.1 Extracellular Polymeric Substances......................... 273
viii Contents

8.2 Soluble Microbial Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

8.3 Steady-State Model Including EPS and SMP . . . . . . . . . . . . . . . . . 276
8.4 Relating EPS and SMP to Aggregate Parameters.............. 278
8.5 Nutrient-Uptake and Acceptor-Utilization Rates . . . . . . . . . . . . . 278
8.6 Parameter Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
8.7 Modeling EPS, SMP, and X1n for a Biofilm Process . . . . . . . . . . . . 283
8.8 Intracellular Storage Products (ISP). . . . . . . . . . . . . . . . . . . . . . . . . 285
8.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
8.10 Problems................................................ 288
9 Reactor Characteristics and Kinetics . . . . . . • • . . . . . . .. .. . . . . . .. • . . . 291
9 .1 Reactor Types. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
9.1.1 Suspended-Growth Reactors......................... 292
9.1.2 Biofilm Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
9 .1.3 Membrane Bioreactors (MBRs). . . . . . . . . . . . . . . . . . . . . . . 296
9.1.4 Biofilm Reactors with Active Substrata. . . . . . . . . . . . . . . . 302
9.1.5 Reactor Arrangements...................... ........ 302
9.2 Important Factors in the Engineering Design of Reactors . . . . . . 303
9.2.1 Selecting an Appropriate SF for Design . . . . . . . . . . . . . . . 304
9.2.2 Effect of SF on System Efficiency for Simple Substrates . . 306
9.2.3 Design When Biosolids Settling or Other Factors
Are Critical. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
9.3 Mass Balances................................... ........ 308
9 .3.1 Batch Reactor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
9.3.2 Continuous-Flow Stirred-Tank Reactor with
Effluent Recycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
9.3.3 Plug-Flow Reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
9.3.4 Plug-Flow Reactor with Effluent Recycle . . . . . . . . . . . . . . 314
9.3.5 Plug-Flow Reactor with Settling and Cell Recycle. . . . . . . 316
9.4 Alternative Rate Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
9.5 Linking Stoichiometric and Mass Balance Equations.......... 318
9.6 Reactors in Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
9.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
9.8 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
9.9 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
10 Meth.anogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
10.1 Uses of Methanogenic Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . 335
10.2 Treating Dilute Wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
10.2.1 The UASB and AFMB.............................. 339
10.2.2 Anaerobic Membrane Bioreactors . . . . . . . . . . . . . . . . . . . 341
10.3 Reactor Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
10.4 Process Chemistry and Microbiology . . . . . . . . . . . . . . . . . . . . . . . 346
10.4.1 Process Microbiology...................... ........ 347
10.4.2 Process Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
10.5 Process Kinetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
10.5.1 Temperature Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
10.5.2 Reaction Kinetics for a CSTR. . . . . . . . . . . . . . . . . . . . . . . . 371
 Contents ix

10.5.3 Complex Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

10.5.4 Process Optimization.............................. 378
10.5.5 Reaction Kinetics for Biofilm Processes............... 380
10.5.6 Kinetics with Hydrolysis as Limiting Factor . . . . . . . . . . 381
10.6 Special Factors in the Design of Anaerobic Biosolids Digesters . 386
10.6.1 Loading Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
10.6.2 Mixing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
10.6.3 Heating. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
10.6.4 Gas Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
10.6.5 Performance...................................... 389
10.7 Example Designs for Anaerobic Treatment of Dilute Wastewater . . 389
10.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
10.9 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
11 Aerobic Suspended-Growth Processes. . . . . . • . . . . . . . . • . . . . . . . . • . . 401
11.1 Characteristics of Classical Activated Sludge. . . . . . . . . . . . . . . . . 402
11.1.1 The Basic Activated Sludge Configuration. . . . . . . . . . . . 402
11.1.2 Microbial Ecology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
11.1.3 Oxygen and Nutrient Requirements . . . . . . . . . . . . . . . . . 405
11.1.4 Impacts of SRT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
11.2 Process Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
11.2.1 Physical Configurations. . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
11.2.2 Oxygen-Supply Modifications . . . . . . . . . . . . . . . . . . . . . . 413
11.2.3 Loading Modifications....................... .... .. 415
11.3 Design and Operating Criteria............................. 417
11.3.1 Historical Background............................. 417
11.3.2 Food-to-Microorganism Ratio. . . . . . . . . . . . . . . . . . . . . . . 418
11.3.3 Solids Retention lime.............................. 419
11.3.4 Comparison of Loading Factors . . . . . . . . . . . . . . . . . . . . . 421
11.3.5 Mixed-Liquor Suspended Solids, the SVI,
and the Recycle Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
11.4 Aeration Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
11.4.1 Oxygen-Transfer and Mixing Rates............ .... .. 425
11.4.2 Diffused Aeration Systems . . . . . . . . . . . . . . . . . . . . . . . . . 428
11.4.3 Mechanical Aeration Systems. . . . . . . . . . . . . . . . . . . . . . . 429
11.5 Bulking and Other Sludge-Settling Problems. . . . . . . . . . . . . . . . . 430
11.5.1 Bulking Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
11.5.2 Foaming and Scum Control. . . . . . . . . . . . . . . . . . . . . . . . . 434
11.5.3 Rising Sludge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
11.5.4 Dispersed Growth and Pinpoint Floe. . . . . . . . . . . . . . . . . 435
11.5.5 Viscous Bulking. . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . 435
11.5.6 Addition of Polymers....................... ..... .. 435
11.6 Activated Sludge Design and Analysis . . . . . . . . . . . . . . . . . . . . . . 436
11.7 Analysis and Design of Settlers . .. . . . . . . . . . . . . . . . . .. . . . . . . . 443
11.7.1 Activated Sludge Properties . . . . . . . . . . . . . . . . . . . . . . . . 444
11.7.2 Settler Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
x Contents

11.7.3 Loading Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449

11.7.4 Basics of Flux Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
11.7.5 State-Point Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
11.7.6 Connecting the Settler and Aeration Tank . . . . . . . . . . . . 462
11.7.7 Limitations of State-Point Analysis . . . . . . . . . . . . . . . . . . 463
11.8 Membrane Bioreactors (MBRs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
11.9 Integrated Fixed-Film Activated Sludge. . . . . . . . . . . . . . . . . . . . . 464
11.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
11.11 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
11.12 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
12 Aerobic Biofilm Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
12.1 Biofilm Process Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
12.2 Trickling Filters and Biological Towers . . . . . . . . . . . . . . . . . . . . . . 478
12.3 Rotating Biological Contactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
12.4 Granular-Media Filters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
12.5 Fluidized-Bed and Circulating-Bed Biofilm Reactors.. ........ 491
12.6 Hybrid Biofilm/Suspended-Growth Processes . . . . . . . . . . . . . . . 497
12.7 Aerobic Granular-Sludge Processes. . . . . . . . . . . . . . . . . . . . . . . . . 497
12.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
12.9 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
13 Nitrogen Transformation and Recovery . . . • • . . . . . . . • • . . . . . . . • • . . . 501
13.1 Nitrogen Forms, Effects, and Transformations................ 502
13.2 Nitrogen's Transformation Reactions . . . . . . . . . . . . . . . . . . . . . . . 503
13.3 Nitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
13.3.1 Biochemistry, Physiology, and Kinetics of
Nitrifying Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
13.3.2 Common Process Considerations............ ........ 514
13.3.3 Activated Sludge Nitrification: Single-Stage
versus Separate-Stage.............................. 514
13.3.4 Biofilm Nitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
13.3.5 Hybrid Processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
13.3.6 The Role of the Input BODr/TKN Ratio . . . . . . . . . . . . . . 527
13.4 Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
13.4.1 Physiology of Denitrifying Bacteria. . . . . . . . . . . . . . . . . . 528
13.4.2 Denitrification Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530
13.4.3 Comparing the Nitrogen-Removal Systems. . . . . . . . . . . 533
13.5 Range of Nitrification and Denitrification Systems............ 537
13.5.1 Biofilm Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
13.5.2 The Barnard Process for Nitrogen Removal . . . . . . . . . . . 540
13.5.3 Sequencing Batch Reactor . . . . . . . . . . . . . . . . . . . . . . . . . . 541
13.5.4 Side-Stream Anammox Treatment . . . . . . . . . . . . . . . . . . . 542
13.6 Nitrous Oxide Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
13.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
13.8 Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548
 Contents xi

14 Phosphorus Removal and Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

14.1 Normal Phosphorus Uptake into Biomass................... 562
14.2 Precipitation by Metal-Salts Addition to a Biological Process . . . 563
14.3 Enhanced Biological Phosphorus Removal . . . . . . . . . . . . . . . . . . 565
14.4 Phosphorus Recovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
14.4.1 Lack of P Removal Opens Up P Recovery . . . . . . . . . . . . 570
14.4.2 Wastewater as a Direct Source of Fertilizer P . . . . . . . . . . 571
14.4.3 Biomass as a Source of Slow-Release P . . . . . . . . . . . . . . . 571
14.4.4 Selective Adsorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
14.4.5 Struvite Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
14.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
14.6 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
15 Biological Treatment of Drinking Water. . . . • . . . . . . . . • . . . . . . . . • . . . 577
15.1 Why Biological Treatment of Drinking Water?. . . . . . . . . . . . . . . . 577
15.2 Aerobic Biofilm Processes to Eliminate Biological Instability . . . 578
15.2.1 General Characteristics of Aerobic Biofilm Processes... 578
15.2.2 Biodegradable Organic Matter (BOM). . . . . . . . . . . . . . . . 579
15.2.3 Inorganic Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
15.2.4 Hybrid Biofiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
15.2.5 Biofilm Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
15.2.6 Slow Biofiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
15.2.7 Release of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . 587
15.2.8 Biodegradation of Specific Organic Compounds. . . . . . . 588
15.3 Anaerobic Biofilm Processes to Reduce Oxidized Contaminants 589
15.3.1 Oxidized Contaminants............................ 589
15.3.2 General Characteristics of Biofilm Processes to Reduce
Oxidized Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
15.3.3 Autotrophic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
15.3.4 Heterotrophic Processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
15.4 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
15.5 Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
A Free Energies of Formation for Various Chemical Species, 25°C..... 603
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611

The following electronic "bonus" Chapters Bl to B5 can be found at www

.mhprofessional.com/ rittmann2e.
B1 Lagoons and Wetlands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B1.1
Bl.1 Aerated Lagoons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.l
Bl.2 Stabilization Lagoons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.5
Bl.3 Types of Stabilization Lagoons . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.6
Bl.4 Aerobic Stabilization Lagoons . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.6
Bl.4.1 Basic Relationships ............................. Bl.7
xii Contents

Bl.4.2 Solar Energy Input and Utilization Efficiency . . . Bl.9

Bl.4.3 BODL Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.11
Bl.4.4 Kinetics of Phototrophic Growth ............. Bl.15
Bl.5 Facultative Stabilization Lagoons . . . . . . . . . . . . . . . . . . . . . . Bl.16
Bl.5.1 BOD5 Surface-Loading Rates . . . . . . . . . . . . . . . . . Bl.17
Bl.5.2 First-Order Kinetics . . .. . . . . . . . . . . . . . . . . .. . . . Bl.18
Bl.6 Anaerobic Lagoons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.21
Bl.7 Series Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.22
Bl.8 Coliform Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.23
Bl.9 Details of Lagoon Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.26
Bl.10 Removing Suspended Solids from the Lagoon Effluent . . . Bl.26
Bl.11 Wetlands Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.27
Bl.12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.29
Bl.13 Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bl.30
B2 Microbiological Detoxification................................ B2.1
B2.1 Factors Causing Molecular Recalcitrance . . . . . . . . . . . . . . . B2.3
B2. l.1 Molecular Structure . . . . . . . . . . . . . . . . . . . . . . . . . B2.3
B2. l.2 Environmental Conditions . . . . . . . . . . . . . . . . . . . B2.3
B2.1.3 Microorganism Presence . . . . . . . . . . . . . . . . . . . . . B2.6
B2.2 Classes of Synthetic Organic Chemicals . . . . . . . . . . . . . . . . . B2.6
B2.3 Energy Metabolism versus Co-metabolism . . . . . . . . . . . . . B2.10
B2.4 Electron Donor versus Electron Acceptor . . . . . . . . . . . . . . . B2.11
B2.5 Minimum Substrate Concentration (Smrn) . . . . . . . . . . . . . . . B2.13
B2.6 Biodegradation of Important Classes of Environmental
Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.15
B2.6.l Hydrocarbons................................ B2.15
B2.6.2 BTEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.17
B2.6.3 MTBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.20
B2.6.4 Polycyclic Aromatic Hydrocarbons (PAHs) . . . . . B2.20
B2.6.5 Chlorinated Solvents and Other Halogenated
Aliphatic Hydrocarbons . . . . . . . . . . . . . . . . . . . . . B2.22
B2.6.6 Chlorinated Aromatic Hydrocarbons . . . . . . . . . . B2.30
B2.6.7 Pentachlorophenol . . . . . . . . . . . . . . . . . . . . . . . . . . B2.32
B2.6.8 Dioxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.33
B2.6.9 Energetics (Explosives) . . . . . . . . . . . . . . . . . . . . . . B2.34
B2.6.10 Synthetic Detergents . . . . . . . . . . . . . . . . . . . . . . . . B2.35
B2.6.11 Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.37
B2.6.12 1,4-Dioxane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.39
B2.6.13 Triclosan and Triclocarban . . . . . . . . . . . . . . . . . . . B2.39
B2.6.14 Perfluorinated Alkanoates . . . . . . . . . . . . . . . . . . . B2.40
B2.7 General Fate Modeling for Organic Chemicals . . . . . . . . . . B2.41
B2.8 Inorganic Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.43
B2.9 In Situ Bioremediation . . . . . . . . .. .. . . . . . .. . . . . . . .. . . . . B2.46
B2.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.48
B2.11 Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.54
 Ca nt ent s xiii

B3 Microbial Electrochemical Cells. . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • B3.1

B3.1 What Is a Microbial Electrochemical Cell? . . . . . . . . . . . . . . . . B3.1
B3.2 Anode-Respiring Bacteria (ARB) and Extracellular Electron
Transport (EET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.5
B3.3 Biofilm-Anode Kinetics and Ecology . . . . . . . . . . . . . . . . . . . B3.7
B3.3.1 Nemst-Monod Kinetics. . . . . . . . . . . . . . . . . . . . . . . . . B3.7
B3.3.2 Multiple EET Pathways and EKA Values . . . . . . . . . B3.9
B3.3.3 Microbial Ecology of the Biofilm Anode . . . . . . . . . B3.10
B3.4 Proton Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.12
B3.5 Mem.branes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.13
B3.6 Cathode Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.14
B3.6.1 Four-Electron Reduction of 0 2-The MFC . . . . . . . B3.15
B3.6.2 Two-Electron Reduction of 0 2-The MPPC . . . . . . B3.15
B3.6.3 Two-Electron Reduction of H 2 0--The MEC B3.16
B3.6.4 Reductions of C02 to Organic Products--
The MESC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.16
B3.6.5 Reduction of Oxidized Contaminants . . . . . . . . . . . B3.17
B3.7 Energy Balance and Over-Potentials . . . . . . . . . . . . . . . . . . . B3.17
B3.7.1 Anode Over-Potential . . . . . . . . . . . . . . . . . . . . . . . . B3.18
B3.7.2 Cathode Over-Potential . . . . . . . . . . . . . . . . . . . . . . . B3.18
B3.7.3 Ohmic Over-Potential . . . . . . . . . . . . . . . . . . . . . . . . B3.19
B3.7.4 Concentration and pH Over-Potentials . . . . . . . . . . B3.19
B3.7.5 Total Over-Potential, Net Output, and Applied
Voltage....................................... B3.20
B3.8 Performance Metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.21
B3.9 MxC Designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.24
B3.9.1 Bench Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.24
B3.9.2 Scaling Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.25
B3.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.27
B3.11 Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.28
B4 Photosynthetic Biofactories. . . . . . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • B4.1
B4.1 Basics of Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.1
B4.1.1 Capturing and Utilizing Light Energy . . . . . . . . . . . . B4.1
B4.1.2 C02 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.3
B4. l.3 Other Forms of Photosynthesis . . . . . . . . . . . . . . . . B4.3
B4.2 Kinetic Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.3
B4.2.1 Light Limitation .. . .. . .. .. .. .. . .. .. .. .. . .. .. . B4.3
B4.2.2 C1 Limitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.6
B4.2.3 Pi Limitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.6
B4.2.4 N; Limitation and TAG Storage . . . . . . . . . . . . . . . . B4.7
B4.2.5 Multiple Limiting Factors . . . . . . . . . . . . . . . . . . . . . B4.8
B4.2.6 Productivities and Nutrient Demands . . . . . . . . . . B4.8
B4.3 Microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.10
B4.3.l Cyanobacteria versus Eukaryotic Algae. . . . . . . . . . . B4.10
B4.3.2 TAG versus DAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.ll
xiv Contents

B4.3.3 TAG Storage and N Depletion ................. . B4.13

B4.3.4 Genetic Modifications of Cyanobacteria ......... . B4.13
B4.3.5 Coccolithophores ............................ . B4.13
B4.3.6 Other Microorganisms ........................ . B4.13
B4.3.7 Extremophiles ...................... ......... . B4.15
B4.4 Reactor Systems ..................................... . B4.16
B4.4.l Open Raceways ............................. . B4.16
B4.4.2 Enclosed Photobioreactors ........... ......... . B4.17
B4.4.3 Productivities B4.17
B4.5 Biomass Harvesting ................................. . B4.19
B4.6 Downstream Processing and the Biorefinery ... ......... . B4.20
B4.6.l Dewatering ................................. . B4.20
B4.6.2 Microbiological Conversion ................... . B4.20
B4.6.3 Extraction .......................... ......... . B4.22
B4.6.4 Transesterification ........................... . B4.22
B4.6.5 Hydrothermal Liquefaction ................... . B4.22
B4.6.6 Nutrient Recovery .................. ......... . B4.22
B4.7 Wastewater Treatment ............................... . B4.23
B4.8 References .......................................... . B4.25
B4.9 Problems .................................. ......... . B4.28
BS Complex Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.1
B5.1 Nonsteady-State Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.1
BS.1.1 Examples of Nonsteady-State Systems . . . . . . . . . . . BS.3
BS.2 Multispecies Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BS.18
B5.2.1 Basic Multispecies Model . . . . . . . . . . . . . . . . . . . . . . BS.19
BS.2.2 Adding the Consumption of the Electron
Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BS.21
B5.2.3 Production of N0-3-N . . . . . . . . . . . . . . . . . . . . . . . . . . BS.23
BS.2.4 Production and Consumption of SMP . . . . . . . . . . . BS.23
BS.2.5 Sequencing Batch Reactor . . . . . . . . . . . . . . . . . . . . . . BS.26
B5.2.6 Multispecies Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . BS.27
BS.2.7 Modeling Multispecies Biofilms . . . . . . . . . . . . . . . . . BS.30
BS.3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BS.33
 Preface
nvirorunental biotechnology utilizes microorganisms to improve the sustainabil-

E ity of human society. These improvements include preventing the discharge of

pollutants to the environment, cleaning up contaminated envirorunents, generat-
ing valuable resources for human society, and improving human health. Envirorunental
biotechnology is essential to society and truly unique as a technical discipline.
Environmental biotechnology is historic and eminently modern. Microbiological
treatment technologies developed at the beginning of the twentieth century, such as
activated sludge and anaerobic digestion, remain mainstays today. At the same time,
new technologies constantly are being introduced to address very contemporary prob-
lems, such as detoxification of hazardous chemicals and recovery of valuable resources.
Important tools used to characterize and control processes in environmental biotech-
nology also span the decades. For example, traditional measures of biomass, such as
volatile suspended solids, have not lost their relevance, even though tools from molecu-
lar biology allow us to explore the diversity of the microbial communities.
Processes in environmental biotechnology work according to well-established prin-
ciples of microbiology and engineering, but application of those principles normally
requires some degree of empiricism. Although not a substitute for principles, empiri-
cism must be embraced, because the materials treated with environmental biotechnol-
ogy are inherently complex and varying in time and space.
The principles of engineering lead to quantitative tools, while the principles of
microbiology often are more observational. Quantification is essential if processes are
to be reliable and cost-effective. However, the complexity of the microbial communi-
ties involved in environmental biotechnology often is beyond quantitative description;
unquantifiable observations are of the utmost value, too.
In Environmental Biotechnology: Principles and Applications, we connect these differ-
ent facets of environmental biotechnology. Our strategy is to develop the basic concepts
and quantitative tools in the first nine chapters, which comprise the principles part
of this second edition. We consistently call upon those principles as we describe the
applications in Chapters 10 through 15, as well as in five "bonus" chapters available
electronically. Our theme is that all microbiological processes behave in ways that are under-
standable, predictable, and unified. At the same time, each application has its own special
features that must be understood. The special features do not overturn or sidestep the
common principles. Instead, they complement the principles and are most profitably
understood in the light of the principles.

xv
xvi Preface

This book is targeted for graduate-level courses in curricula that exploit microbio-
logical processes for environmental quality control The book also is appropriate as a
text for upper-level undergraduate courses and as a comprehensive resource for those
engaged in professional practice and research involving environmental biotechnology.
The material in this second edition of Environmental Biotechnology can be used in one
or several courses. For students not already having a solid background in microbiology,
Chapters 1 to 4 provide a foundation in taxonomy, metabolism, genetics, and ecology.
These chapters address the microbiology concepts that are most essential for under-
standing the principles and the applications that follow. They can serve as the text for
a first course in environmental microbiology, or they can be used as a resource for stu-
dents who need to refresh their knowledge in preparation for a more process-oriented
course, research, or practice.
Chapters 5 through 9 provide the quantitative core of the principles. Chapter 5 devel-
ops quantitative tools for describing the stoichiometry and energetics of microbial reac-
tions: what and how much the microorganisms consume and produce. Stoichiometry is
the most fundamental of the quantitative tools. Chapters 6 and 7 systematically develop
quantitative tools for kinetics: how fast are materials consumed and produced. Chapter 6
is for suspended-growth processes, while Chapter 7 is for biofilm processes. Described
in Chapter 8 are some of the products that microorganisms make that affect process
performance and ways to quantify them. The understanding expands the systematic
tools of Chapters 6 and 7. Reliability and cost-effectiveness depend on applying kinet-
ics properly. Chapter 9 describes how principles of mass balance and kinetics are used
to apply stoichiometry and kinetics to the range of reactors used in environmental
practice.
Chapters 10 through 15 comprise the applications section of the second edition.
Each chapter includes information on the stoichiometry and kinetics of the key micro-
organisms, as well as features that are not easily captured by stoichiometric or kinetic
parameters. Each chapter explains how processes are configured to achieve treatment
objectives and what are the quantitative criteria for a good design. The objective is to
link principles to practice as directly as possible.
We have reorganized the applications part to emphasize our goal of using envi-
ronmental biotechnology to improve the sustainability of human society. Most promi-
nently, we have made the first chapter of the applications part about methanogenesis,
since it is the primary means to convert organic pollutants into a valuable energy form,
methane gas. Methanogenic treatment can turn wastewater treatment into a net genera-
tor of renewable energy, instead of a major energy consumer.
Chapters 11 and 12 delve into the wide range of aerobic treatment processes for
treating wastewater to remove biochemical oxygen demand (BOD). These processes
are used worldwide and must be understood deeply to ensure that they perform well.
Chapters 13 and 14 address the transformations, removal, and/or recovery of nitrogen
and phosphorus. Many new developments have occurred in these areas since the first
edition of Environmental Biotechnology was published. We describe the new advance-
ments in science and technology in the second edition, and we give special attention
to recovering valuable resources in wastewater while reducing the energy required to
do so, rather than just removing polluting materials from wastewater, as commonly
done in the past. Chapter 15 describes the use of biofilm processes to prepare safe and
palatable drinking water, a topic whose acceptance has increased greatly since the first
edition was published.
 Preface xvii

Those who have purchased the print textbook from McGraw-Hill also will gain
access to five electronic "bonus" chapters: Chapter 81, Lagoons and Wetlands; Chapter
82, Microbiological Detoxification; Chapter 83, Microbial Electrochemical Cells; Chapter
84, Photosynthetic Biofactories; and Chapter 85, Complex Systems. These chapters can
be found at www.mhprofessional.com/rittmann2e. They are being published electroni-
cally in order to lower the length and cost of the print book. That the bonus chapters are
available only in electronic versions does not mean that they are of less importance. In
fact, Chapters 82, 83, and B4 present information on some of the hottest new topics in
environmental biotechnology. The bonus chapters are included in the ebook of this text.
One important feature of Environmental Biotechnology is that it contains many
examples. These examples illustrate the step-by-step procedures for utilizing the tools
needed to understand how microbial systems work or to design a treatment process. In
most cases, learning by example is the most effective approach, and we give it strong
emphasis.
The book also has extensive sets of problems at the ends of its chapters. The prob-
lems can be assigned as "homework," used as supplemental examples in class, or
examined as study tools.
In an effort to promote uniformity in notation, we have elected to adapt the
"Recommended Notation for Use in the Description of Biological Wastewater Treatment
Processes," agreed upon internationally and as published in Water Research 16,
pp. 1501-1505 (1982). We hope this will encourage others to do the same, as this will
facilitate much better communication among us.
We take this opportunity to thank our many wonderful students and colleagues,
who have taught us new ideas, inspired us to look farther and deeper, and corrected
our frequent errors. The numbers are too many to list by name, but you know who you
are.
Finally, we thank Marylee and Martha for loving us, even when we became too
preoccupied with the ''book project."

Bruce E. Rittmann
Tempe, Arizona
Perry L. McCarty
Stanford, California
Environmental
Biotechnology
 CHAPTER 1
Moving Toward
Sustainability
n the 1970s, when the U.S. Environmental Protection Agency was first formed, the

I major environmental concern for water was pollution of rivers, streams, and lakes.
Other major concerns were with air and land contamination. Since then, the world's
population has more than doubled, and the availability of some of the world's natural
resources (including water) to support human activities is shrinking toward unsustain-
able levels. Additionally, the release of greenhouse gases from the combustion of fossil
fuels and from other sources has caused the Earth's temperature to rise, causing a host
of significant and urgent environmental problems (IPCC, 2018; NAS, 2018; Reidmiller
et al., 2018; U.S. EPA, 2018). Among the obvious are extraordinary storms and floods
in some locations, greater droughts and extreme fires in others, melting of glaciers, a
constant rise in sea levels, and the acidification of the oceans with an accompanying
loss of sea life. These major problems, which have come to the fore relatively recently,
require urgent attention and effective action. As environmental engineers and scientists,
we must perform well our job of water-quality protection, but we must also address
the growing need to preserve resources and reduce greenhouse gases as effectively as
possible.
Environmental 'biotechnology is one of the powerful tools that we have for addressing
the emerging and the long-standing challenges to sustainability. Environmental bio-
technology can be defined as "managing microbial communities so that they provide
services to society." The services include removing pollutants from water and other
contaminated media, generating renewable resources, and improving human health. It
is obvious that such services can address many of the pressing challenges facing human
society. In Chapter 1, we begin by providing a framework for understanding the chal-
lenges that environmental engineers and scientists face, along with the opportunities
for environmental biotechnology.

1.1 Water Uses and Resources

Beneficial uses of water require that the water have proper quality. Only a small portion
of the total water available in the world has the quality suitable for most human needs,
and difficulties are experienced in keeping the usable water free from contamination by
human or industrial wastes. Environmental engineers have been given the responsibil-
ity to protect, store, and transport water; preserve its quality for aesthetic and environ-
mental needs; and upgrade its quality for personal, agricultural, and industrial uses.
These responsibilities present significant challenges.

1
2 Chapter One

Table 1.1 contains a summary of the global availability of water. Most of the world's
water is contained in the ocean, where the salt content is about 3.4%, much too high for
most human uses, including irrigation, drinking, and washing. The largest portion of
freshwater, which makes up only 2.5% of the total, is locked in polar ice and glaciers.
Liquid freshwater, the only water readily available with adequate quality to satisfy our
greatest needs, represents less than 1% of the total water on Earth. In addition, most of
this lies deep in the ground where it is difficult, if not impossible, to obtain.
While water is a renewable resource, our use of available freshwater is already high
and growing so rapidly that it could exceed its rate of renewal in coming years. Solar
radiation continually evaporates (mostly) seawater, leaving the salt and other chemi-
cals behind. A portion of the resulting water vapor eventually condenses and falls on
land as rain or snow to renew the supply of fresh and relatively clean water. It is the
total rainfall of 119,000 km3 /year, rather than the Earth's total freshwater reservoir, that
actually represents the sustainable supply, since it is new water each year. However,
not all rainfall can be used. About two-thirds (74,200 km3) evaporates before it can be
captured; of the remainder that is stored or runs off the land (44,800 km3), only about
one-half can practically be captured for use.

1.2 Wastewater's Resources

Water is by far our most widely used natural resource. On a mass basis, water is used
by the world's population at a rate more than 100-fold greater than for all other natural
resources combined. Not only do we use water for drinking and washing, but we use
it for agriculture to grow crops we eat, to generate electrical power and other forms of
energy that run our society, for industry to produce the goods we use, for a broad range
of municipal and commercial purposes, and for ecosystem preservation. Furthermore,
water is used to carry away some of our wastes.
After we have used and dirtied water, we have commonly thought of it as waste-
water, a term that is used throughout this book. Sewage is a term used to describe

TABLE 1.1 World water Budget

water Type and Location Amount, km* Percentage of World's Total Water
Total water 1,386,000,000 100.0
Salt water 1,350,000,000 97.5
Freshwater 34,600,000 2.5
Ice 23,800,000 1.7
Liquid 10,800,000 0.8
Groundwater* 10,400,000 0.75
Surface water 90,000 0.007
Water vapor (atmosphere) 13,000 0.001
Yearly rainfall on land 119,000 -

•About one-half of the groundwater lies greater than 1.5 kilometers below the ground surface.
Source: Based upon information from Shiklomanov (1998).
 Moving Toward Sustalnablllty 3

wastewater coming directly from humans. Ironically, what we call wastewater is water
that contains a wide range of resources, which we could term to be "used resources."
For this reason, what was once viewed as waste is now being looked at as a resource,
one that must not be wasted, but cleansed, captured in useful form, and used once
again. For example, Singapore no longer uses the term wastewater; instead, they call it
"used water." The needed degree of treatment for used water depends upon the pur-
pose for which it is to be used. This is called "fit for use" treatment (Li et al., 2015).
Wastewaters contain important resources that can be captured and used to satisfy
the basic needs of human society. The most important resource often is the water itself,
once it has been cleaned to prevent harm to the consumer using fit-for-use treatment.
Many wastewaters contain organic matter that contains energy, which, if captured, can
be used to run our treatment systems and be sold on the market to generate income
(Rittmann, 2013). Other commonly present resources are the fertilizing elements, nitro-
gen and phosphorus. Phosphorus is present in wastewater as simple phosphate or
complex organic phosphate. H the simple phosphate is captured in a correct form, it
can be removed, concentrated, and used once again as fertilizer (Rittmann et al., 2011).
Nitrogen, generally in the form of ammonium or organic nitrogen in wastewater, also
can be recovered and reused in agriculture. One key for reusing the phosphorus and
nitrogen for agriculture is ensuring that treatment and recovery eliminate pathogens.
A second key is that the recovered nutrient be in a form that can be available as a plant
nutrient.
Important to realize is that, in order to feed the world's growing population, N 2 is
now taken from the atmosphere and converted into ammonium for use as a crop fertil-
izer. The Haber-Bosch process, used for N 2 fixation to ammonium, consumes about
7% of the world's natural gas, one of the fossil fuels causing climate change (McCarty
et al., 2011). The nitrogen in municipal wastewater comes from the food we eat, and the
energy originally used to obtain that nitrogen from atmospheric N 2 represents as much
energy as we currently use to run an aerobic wastewater treatment system. However,
traditional wastewater treatment for nitrogen removal generally converts the ammo-
nium back into N 2 gas. But instead of converting it back to N2' we could reduce overall
fossil fuel energy consumption if we recovered and used the ammonium directly as
fertilizer.
A similar analysis can be made for phosphorus. Today, almost all phosphate is
mined and used in agriculture. Most of that phosphate ends up in waterways due to
run-off and wastewater discharges (Rittmann et al., 2011); this "lost phosphorus" spurs
eutrophication and hypoxia. Traditional phosphate removal generates inorganic solids
that are not usable in agriculture; that phosphate cannot be reused where it is needed.
The greatest sustainability benefit comes from removing phosphate in a form that is
readily useful in agriculture.

1.3 Climate Change

The processes used for transporting and cleaning water use energy, and this energy often
comes from fossil-fuel combustion, thus adding to the rising problems from climate
change. While the energy used for water represents only a modest fraction of the world's
total fossil fuel use, it is a fraction that environmental engineers and scientists can address
by finding ways to lower the amount of energy used for water transportation and for
water and wastewater treatment.
4 Chapter One

Fossil-fuel combustion is not the only water-related factor contributing to green-

house gases. Some of the byproducts emanating from treatment processes are green-
house gases. For example, methane gas (CH4 ) produced by anaerobic wastewater
treatment is a good renewable energy resource that can help limit our use of fossil fuels;
however, if CH4 is allowed to escape to the atmosphere, the impact on climate change
is large because methane has a greenhouse-gas warming potential 25 to 30 times that of
C02(U.S. EPA, 2018). Methane emissions from sewers and sanitary landfills, for which
we are responsible, must also be reduced.
Another greenhouse gas that is produced through biological wastewater treatment
is nitrous oxide (Np), and it has a warming potential nearly 300 times greater than
that of carbon dioxide. If just a small fraction of the nitrogen entering wastewater
treatment facilities is converted to Np and escapes to the atmosphere, efforts to
reduce fossil fuel use would be overshadowed by the impact of the formation and
release of N 20.
In 2016, the total human global emission of greenhouse gases was estimated to be
42 billion tons of C02equivalents (IPCC, 2018). The United States contributed 6.5 billion
tons, or more than 15% of that (U.S. EPA, 2018). Of the U.S. contribution, 82% came from
C02 emissions. While worldwide CH4 and N 20 emissions are relatively small in mass
terms compared with co2, their much larger global-warming potential means that
their emissions in 2016 caused approximately 10% and 6%, respectively, of the overall
climate-change impact.
The several human contributions of greenhouse gases are largely responsible for the
Earth's temperature rise of about 1°C over the last 150 years, and the temperature will
continue to rise if humans do not reduce their emissions of all the radiation-absorbing
chemicals. The International Panel on Climate Change (IPCC, 2018) indicated that
releases of fossil C02will need to go to net zero by 2055, along with net zeroing of non-
C02 greenhouse gases by 2030, if global temperature is not to rise by more than 2°C.
Keeping the temperature rise to less than 2°C will require that greenhouse gases be net
removed from the atmosphere! All economic sectors-worldwide-will need to make
reduction of greenhouse gases a top priority, and this will include sectors involving
environmental engineering and science professionals.

1.4 Sustalnablllty
Today, a biological process must reliably achieve its effluent standards in a cost-effective
manner, and it also must advance our society's sustainability needs for the future.
As stated in the Brundtland Report (Brundtland, 1987) for the World Commission
on Environment and Development, sustainable development "meets the needs
of the present without compromising the ability of future generations to meet their
own needs." The concept of sustainability was posited even earlier-in the 1970 U.S.
National Environmental Policy Act-as a means to "create and maintain conditions,
under which humans and nature can exist in productive harmony, that permit fulfilling
the social, economic, and other requirements of present and future generations."
In general, sustainability represents a goal of balancing efforts to meet human needs
today without destroying the natural environment upon which future generations will
depend. Today, the concept of sustainability involves at least three interdependent
pillars: economic development, social development, and environmental protection.
They constitute the "triple bottom line" that accounts for social, environmental, and
financial benefits and costs.
 Moving Toward Sustalnablllty 5

TABLE1..2 The Three Pillars of Sustainability Together with Topics of Importance under Each as
Defined by the U.S. Environmental Protection Agency

Environ mental Socia I Economic

Ecosystem Services Environmental Justice Jobs
Green Engineering & Human Health Incentives
Chemistry
Air Quality Participation Supply and Demand
Water Quality Education Natural Resource
Accounting
Stress ors Resource Security Costs
Resource Integrity Sustainable Communities Prices

To help advance sustainability goals, in 2015 the U.S. Environmental Protection

Agency produced a Sustainability Primer that defines six broad topics atop each of the
three pillars. The six topics for each pillar are listed in Table 1.2. For example, the envi-
ronmental pillar includes ecosystem services, which emphasize efforts to protect, sustain,
and restore the health of critical natural habitats and ecosystems. Stressors, including
water pollutants and greenhouse gas emissions, are to be reduced. For resource integ-
rity, waste generation is to be reduced to prevent accidental release and future cleanup
liability. Within the social pillar, environmental justice calls for empowering communities
overburdened by pollution to take action to improve their health and environment.
Resource security means to protect, maintain, and restore access to basic resources. For
the economic pillar, supply and demand uses accounting and market practices to promote
environmental health and social prosperity, while natural resource accounting acts to
improve understanding and accounting of ecosystem services using cost-benefit analy-
sis. Costs is the example of striving to develop waste-free processes, thus minimizing the
needs for regulation, treatment, and disposal costs, while prices refer to reducing risks
for new technologies through demonstration and testing with community partners.

1.5 The Role of Environmental Biotechnology

Environmental biotechnology provides means to achieve many of the sustainability
goals for water. Here are a few prominent examples:

• Anaerobic treatment can capture the energy value of organic matter found in
many used waters. This can make the treatment process energy generating,
which saves money for the operator and lowers society's use of fossil energy.
• Anaerobic treatment also converts nitrogen and phosphorus into inorganic
forms (ammonium and phosphate) that can be recovered and used as feedstock
for agricultural fertilizer.
• Anaerobic and aerobic treatment can detoxify harmful chemicals and make the
water safe for various beneficial uses.

Although wastewater contains valuable resources, current treatment practices

often discard them. Today, the choice of a biological treatment process must go beyond
only meeting the immediate effluent standards. Instead, a well-chosen process should
6 Chapter One

accomplish the basic mission of good effluent quality in ways that help achieve the
long-term sustainable needs of society by viewing used waters as holders of resources.
Modifications to existing processes and the development of new processes should be
channeled toward meeting the needs for a sustainable future, an urgent goal for all of
human society.

1.6 Organization of the Book

Attaining all of the good sustainability outcomes requires a firm understanding of the
science and engineering fundamentals underlying environmental biotechnologies.
Applying this understanding to design and operate facilities for improving water
quality treatment is the major emphasis in this book.
Consistent with its title, this book is organized into two parts. The first part, com-
prising Chapters 2 to 9, lays out the principles underlying all microbiological processes:
e.g., biochemistry, ecology, stoichiometry, and kinetics. These principles represent the
essential knowledge for understanding, designing, and operating any environmental
biotechnology.
The second part, Chapters 10 to 15, applies those principles to a wide range of appli-
cations. Consistent with our focus on sustainability, Chapter 10 is on methanogenesis,
in which the energy embodied in organic compounds in used waters is converted to
methane gas, a valuable fuel. Unlike the fossil methane in natural gas, methane coming
from a methanogenic process is renewable and carbon-neutral if captured and used.
Chapters 11 to 15 address aerobic treatment and removal and recovery of nutrients,
including traditional approaches for improving water quality and emerging approaches
for recovering valuable resources.
Those who purchase the textbook from McGraw-Hill also will gain access, at
www.mhprofessional.com/rittmann2e, to five electronic "bonus" chapters: Lagoons
and Wetlands, Microbiological Detoxification, Microbial Electrochemical Cells,
Photosynthetic Biofactories, and Complex Systems. They are being made available in
electronic versions in order to constrain the length and cost of the printed book.

1. 7 References
Brundtland, G. H. (1987). Our Common Future. New York: World Commission on
Environment and Development.
Inter-Governmental Panel on Climate Change (IPCC) (2018). Global Warming of 1.5°C.
Switzerland: IPCC, p. 28.
Li, W.-W.; H.-Q. Yu; and B. E. Rittmann (2015). "Reuse water pollutants." Nature. 528,
pp. 29-31.
McCarty, P. L.; J. Bae; and J. Kim (2011). "Domestic wastewater treatment as a net energy
producer-can this be achieved?" Environ. Sci. Technol. 45, pp. 7100-7106.
NAS (2018). Environmental Engineeringfar the 21st Century: Addressing Grand Challenges.
Washington, DC: National Academies Press, p. 120.
Reidmiller, D. R.; C. W. Avery; D. Barrie; A. Dave; B. DeAngelo; M. Dzaugis; M. Kolian;
K. Lewis; K. Reeves; and D. Wmner (2018). Overview: Impacts, Risks, and Adaptation
in the United States: Fourth National Climate Assessment, Volume II. Washington, DC:
U.S. Government Publishing Office, pp. 33-71.
 Moving Toward Sustalnablllty 7

Rittmann, B. E. (2013). "The energy issues in urban water management." In

T. Larsen; K. Udert; and J. Liener, Eds. Wastewater Management: Source Separation and
Decentralisation. London: IWA Publishing, chap. 2, pp. 13-28.
Rittmann, B. E.; B. Mayer; P. Westerhoff; and M. Edwards (2011). "Capturing the lost
phosphorus." Chemosphere. 84, pp. 846--853.
Shildomanov, I. A. (1998). World Water Resources: A New Appraisal and Assessment for the
21st Century. Paris: United Nations Educational, Scientific and Cultural Organization.
U.S. EPA (2018). Inventory of U.S. Greenhouse Gas Emissions and Sinks. Washington, DC:
U.S. Environmental Protection Agency, p. 655.
 CHAPTER 2
Basics of Microbiology

nvironmental biotechnology applies the principles of microbiology to the solu-

E tion of environmental problems. Applications in environmental biotechnology

include the following:
• Treatment and resource recovery from industrial, municipal, and domestic
wastewaters
• Enhancement of the quality of drinking water
• Restoration of environmental soil, water, or air contaminated with undesirable
compounds
• Protection and restoration of rivers, lakes, estuaries, and coastal waters from
environmental contaminants
• Prevention of exposure by pathogens to humans and other species
• Production of environmentally benign chemicals, fuels, and feedstocks
• hnplementation of sustainable resource recovery and utilization, and disposal
practices
Although this textbook cannot address every topic that can be categorized as "envi-
ronmental biotechnology," the principles that apply in one environmental application
often apply equally well to other applications. What is required in all cases is linking
the principles of microbiology with engineering fundamentals.
This chapter reviews the basic principles of microbiology. It lays the foundation
for the fundamentals of biochemistry, metabolism, genetics and information flow, and
microbial ecology, which are addressed in two subsequent chapters. All of these micro-
biology fundamentals prepare the reader to use the engineering fundamentals laid out
in Chapters 5 through 9. Readers desiring more detailed information on microbiology
fundamentals are referred to texts such as Madigan et al. (2018), Madsen (2016), and
Pepper et al. (2014).
This chapter summarizes the following:
• The nature of microbial cells
• How microorganisms are classified (taxonomy)
• What they look like (morphology)
• Their composition (cellular chemicals)

9
10 Chapter Twa

2.1 The Microbial Cell

The cell is the fundamental building block of life. A cell is an entity that is separate from
other cells and its environment. As a living entity, a cell is a complex chemical system
that can be distinguished from nonliving entities in the four critical ways.

1. Cells are capable of growth and reproduction; that is, they can self-produce
another entity essentially identical to themselves.
2. Cells are highly organized, and they selectively restrict what crosses their
boundaries. Thus, cells are at low entropy compared to their environment.
3. The major elements of which cells are composed are C, H, 0, N, P, and S.
4. Cells are self-feeding. They take up necessary elements, electrons, and energy
from their external environment to create and maintain themselves as organized
and reproducing entities. They require sources of the elemental building blocks
that they use to reproduce themselves. They require a source of energy to fuel the
chemical processes needed to sustain life. And, they require a source of electrons
to reduce their major elements. How the cells obtain elements, energy, and
electrons is called metabolism, and it is an essential way in which we characterize
cells. Understanding metabolism is a theme that runs throughout this book.
Cells are physically organized so that they can carry out the processes that make
them living entities. The distinguishing features of a living cell are as follows:

• Cell membrane: a highly selective permeable barrier between the cell and its
environment. Also called the cytoplasmic membrane, it is the structure that
enables the cell to restrict what crosses its boundaries, as well as being a location
for some of the reactions catalyzed by cells for their metabolism.
• Cell wall: a structural member that confers rigidity and shape to the cell and
protects the membrane.
• Cytoplasm: most of the inside of the cell. It contains water and the macromolecules
that the cell needs to function.
• Chromosome: a structure of nucleic acids (DNA) and protein that stores the
genetic code for the cell's heredity and biochemical functions.
• Ribosomes: structures consisting of ribonucleic acids (RNAs) and proteins that
convert the genetic code into the working catalysts that carry out the cell's
reactions.
• Enzymes: protein catalysts that carry out the cell's necessary biochemical
reactions.

Cells may have other components, but these are the essential ones that define them as
living entities.
Living cells are categorized into three major domains based upon genetic simi-
larities: Bacteria, Archaea, and Eukarya. Individual members of these domains are
designated as bacteria, archaea, or eukaryotes. Figure 2.1 shows the three domains
in a phylogenetic tree. Organisms with close branches on the tree are similar. Distant
branches are phylogenetically distinct. The three domains originated from a single root,
and their genetic divergence is estimated to have occurred close to 4 billion years ago,
 Basics af Mlcroblology 11

Euryan:haeota
Mitochondrion
Green nonsulfur
Gram b et .
positives a ena

Cyanobacteria Plants

F1auRE 2.1. Phylogenetic tree of life as determined from comparative ribosomal RNA sequencing.
(Source: Developed from a figure by Woese et al. (1990) and with modifications from Madigan
et al. (2018).)

with Bacteria branching off from Archaea and Eukarya, which then subsequently
diverged around 2 billion years ago.
Despite phylogenetic differences, Bacteria and the Archaea share the physical char-
acteristic of lacking a membrane-enclosed nucleus and, hence, are referred to collec-
tively as prokaryotes (from the Greek for before nucleus). Consequently, the chromosomes
of prokaryotes reside in the cytoplasm. In contrast, chromosomes of Eukarya (or Greek
for true nucleus) exist in a membrane-enclosed nucleus that is distinct from the cyto-
plasm. Furthermore, eukaryotic cells tend to be much larger and structurally more com-
plex than prokaryotic cells.
All three domains include single-celled organisms, while larger multicellular
organisms, including all higher plants and animals, are solely eukaryotic. Single-celled
microorganisms are individual living entities, while the cells of multicellular organisms
cannot live and grow independently, but exist only as part of a multicellular entity.
Some cells may undergo change in form or function through the process of differen-
tiation. For example, all cells within the human body contain the same genetic informa-
tion, but act differently depending upon whether they form part of an eye, a muscle,
or a strand of hair. Cells often can interact with one another through various chemical
signals in ways that can change their form or function as part of the process of differen-
tiation. Of great importance also is that cells can evolve into organisms that are markedly
different from the parent, a process that usually is quite slow, but nevertheless of great
importance to the formation of new organisms or to the development of new capabili-
ties that may aid in organism survival.
While our interest in environmental biotechnology centers primarily on the single-
celled organisms, many of which are from the Bacteria and Archaea domains, we need
to be knowledgeable about organisms in the Eukarya domain as well. Microorganisms
of particular interest here are algae and protozoa, but plants can also be of importance.
12 Chapter Twa

For example, phytoremediation is a process by which plants help to bring about the
destruction of toxic chemicals in soils and groundwater. Here, trees such as the poplar
take up toxic chemicals along with water; in some cases, trees can even transform the
toxic compounds into non-harmful products.

2.2 Microbial Classification

Due to the vast diversity of microorganisms and their wide-ranging functions, scientists
and engineers classify them according to a variety of criteria. Classification that is based
upon observable physical/ chemical cellular properties is called phenotypic classification
and may involve a cell's structure (e.g., morphology, presence of organelles); type of
metabolism {e.g., phototroph (light-eater), lithotroph (earth-eater), aerobe (oxygen-
user), or methanogen (methane-generator)]; preferred environmental condition [e.g.,
thermophile (heat-loving), halophile (salt-loving)]; behavior (e.g., motility, attachment,
fruiting bodies); the manner in which it interacts with dyes or staining (gram positive,
gram negative, acid fast); and the types of lipids it has in its cell membrane. Table 2.1
summarizes many of the phenotypic features that distinguish among Bacteria, Archaea,
and Eukarya. For example, only Eukarya have a membrane-enclosed nucleus, while
only Archaea are capable of generating methane gas (methanogenesis).
A second type of classification for cells is called phylogenic classification, which cat-
egorizes organisms according to the genetic characteristics (genotype) encoded in the
organism's DNA (deoxyribonucleic acid). Although phylogenetic classification can
involve comparisons of a cell's entire chromosome (all of its DNA), most phylogeny is

TABLE 2.1. Differentiating Phenotypic Features among Bacteria, Archaea, and Eukarya

Characterl•tlc Bacteria Archaea Eukarya

Membran&enclosed nucleus Absent Absent Present
Cell wall Muramic acid Muramic acid Muramic acid
present absent absent
Chlorophyll-based photosynthesis Yes No Yes
Methanogenesis No Yes No
Reduction of S to H2S Yes Yes No
Nitrification Yes Yes No
Denitrification Yes Yes No
Nitrogen fixation Yes Yes No

Synthesis of poly-,B- Yes Yes No

hydroxyalkanoate carbon storage
granules
Sensitivity to chloramphenicol, Yes No No
streptomycin, and kanamycin
Ribosome sensitivity to No Yes Yes
diphtheria toxin

Source: Based on Madigan et al. (2018) with modification.

 Basics af Mlcroblology 13

focused on one component of the ribosomal RNA gene (the 165 for Bacteria and Arch.aea
and the18S for Eukarya). The smaller component is utilized for two main reasons. First,
the DNA of every known organism contains a gene that encodes ribosomal RNA.
Second, the RNA gene has conserved (always the same) and variable regions, making it
a good target for grouping and differentiating cells, as needed (Olsen and Woese, 1993).
An added benefit of using the RNA gene is that we currently have sequenced the entire
genome (containing millions of base pairs) for only a few thousand microorganisms,
while we have sequenced the ribosomal DNA sequences (containing thousands of base
pairs) for millions of microorganisms.
An interesting historical fact about phylogenetic sequencing is that microbiologist
Carl R. Woese used this then-nascent method, which he pioneered in the late 1970s,
to demonstrate that the large group of single-celled microorganisms, once collectively
termed bacteria, is actually comprised of two very distinct domains, Bacteria and
Archaea. This was a revolutionary concept at the time, and now it is well accepted.
Classification by phenotype relates organisms based on observable or functional
characteristics, while phylogeny relates organisms based upon their evolutionary his-
tory. Both are powerful methods of classification, and both yield different and comple-
mentary kinds of information. Environmental biotechnology depends on both types of
classification.
Regardless of the classification method chosen, basic taxonomy (science of classifica-
tion), which is used to name, describe, and categorize microorganisms, utilizes com-
mon naming conventions. The taxonomic hierarchy formally contains seven levels,
from domain to species, which are summarized in Table 2.2. The basic taxonomic unit is
the species, which is the collection of strains having sufficiently similar characteristics to
warrant being grouped together. Such a loose definition often makes it difficult to deter-
mine the difference between strains (members of a given species that have measurable
differences between themselves) and species. Groups of species with major similarities
are placed in collections called genera (or genus for singular), groups of genera with suf-
ficient similarity are collected into families, and so on up through domains.
The convention in microbiology is to employ the binomial naming system, with
genus and species names applied for a collection of microorganisms having sufficiently
similar characteristics to warrant being grouped together. Genus names can be abbrevi-
ated (species names cannot), and both are italicized (e.g., Escherichia coli or E.coli and
Methylosinus trichosporium or M. trichosporium). (If italics are not possible, the genus and
species names are underlined.) Closely related microorganisms that are of the same

TABLE 2.2 Taxonomic Naming Hierarchy Applied to the Three Domains of Life

Tuon Example 1 Example 2 Example 3

Domain Bacteria Arch aea Eukarya
Phylum Proteobacteria Euryarchaeota Ascomycota
Class 1-Proteobacteria Methanobacteria Saccharomycetes

Order Enterobacteriales Methanobacteria les Saccharomyceta les

Family Enterobacteriaceae Methanobacteriaceae Saccharomycetaceae
Genus Escherichia Methanobacterium Saccharomyces
Species coli congolense cerevisiae
14 Chapter Twa

species, but have sufficient differences to warrant different designations, are given
strain names, and these are not abbreviated or italicized (e.g. M. trichosporium Ob3B).
A formal compilation of taxonomy for microorganisms is published in Bergey's
Manual of Systematics of Archaea and Bacteria by Wiley Online Library since 2015. It
contains in some detail the individual differences among the major groupings of
microorganisms.

2.3 Prokaryotes
The prokaryotes, Bacteria and Archaea, generally are found together and often par-
ticipate together to bring about the destruction or mineralization of complex organic
or inorganic materials, such as in the formation of methane from the decay of dead
plants and animals or in the oxidation of ammonia to nitrite and nitrate. For example,
within the organic situation, Bacteria ferment and convert complex organic materials
into acetic acid and hydrogen gas, and Archaea convert the acetic acid and hydrogen
into methane gas. The organisms must work closely together, much like an assembly
line, in order to break down the organic matter. With ammonium oxidation to nitrite,
Archaea cannot compete with Bacteria in the oxidization of high ammonia concentra-
tions, such as in wastewater treatment, but can outcompete them when ammonium is
in very low concentration, such as is typical in ocean water. As far as we know today,
only Bacteria have the ability to oxidize nitrite to nitrate.
Another distinction that became clearer with the development of DNA-based
phylogeny is the relationships among the photosynthetic microorganisms. In the
past, algae described the group of single-celled microorganisms that behave like
plants: that is, they contain chlorophyll and derive energy for growth from sunlight.
However, a group of photosynthetic prokaryotes, formerly called blue-green algae, has
no nucleus. Today, they are classified within a bacterial grouping called Cyanobacteria.
Cyanobacteria are Bacteria, and algae are Eukaryotes. Cyanobacteria and algae com-
monly are found together in natural waters and tend to compete for the same energy
and carbon resources. Cyanobacteria are a pesky group of phototrophs that cause many
water quality problems, from tastes and odors in drinking water to the production of
toxins that kill cows and other ruminants that may consume them while drinking from
highly infested surface waters. Despite important differences between algae and cya-
nobacteria, grouping them together makes practical sense when those differences are
not of interest. Here, we are reminded that nature does not strive to classify things;
humans do. Despite many gray areas where classification of microorganisms is not easy
(and sometimes does not seem to make much sense), classification is essential for our
organization of knowledge and for communication among scientists, practitioners, and
others.
Living things cover a very broad spectrum of attributes with a great diversity of
overlapping capabilities. Also, much exchange of genetic information occurs between
species, not only between organisms within a given domain, but also between organ-
isms living in different domains as well. The graying of boundaries that this causes
creates major problems for those seeking the systematic ordering of things. To make
the picture somewhat more complicated, the interest in environmental biotechnology is
often more on function than it is on classification into species. Like cyanobacteria and
algae, which carry out the similar function of photosynthesis, the ability to biodegrade
a given organic chemical may rest with organisms spanning many different genera.
 Basics af Mlcroblology 15

Thus, the identification of a particular species is important in some instances, such as

when concerned with the spread of infectious diseases, but it is not at all of interest to us
in other circumstances. For instance, in a biological wastewater treatment system that
is biodegrading an industrial organic waste efficiently and reliably, the dominant bacte-
rial species may change from day to day. These treatment systems are not pure-culture
systems, and it would be impossible to maintain them as such. They are open, complex,
mixed-culture systems that are ruled by principles of microbial ecology. Species that
can find a niche for themselves and can dominate over their competitors will survive
and flourish, at least until some new chemical or environmental perturbation affects
the system in such a way as to push the balance in some other. Such competition and
change in dominance with changing conditions is one characteristic that allows envi-
ronmental systems to be robust and to work so well.

2.3.1 Bactertal and Archaeal Cell Structure and Function

Bacteria and Archaea are among the smallest of the entities that are generally agreed
to be "living." They range in size from 0.2 µm to around 700 µm in diameter. Their
activities are essential to the survival of all other species due to their key roles in the
basic nutrient cycles and in degrading organic matter. Prokaryotes are ubiquitous in
the biosphere, occupying every geographic niche, from the ocean depths to the highest
mountains, and are distributed throughout the world on air currents and through water
movement. Species diversity is immense, with estimates of 2 to 3 billion microbial spe-
cies, less than 0.5% of which have been identified. Since many functions are distributed
across large phylogenetic distances, even when the same species are not present at dif-
ferent locations, the functions carried out by the present microorganisms can be similar,
driven by environmental selection, such as in the mineralization of dead plants and
animals.
Bacteria and Archaea are important to Nature, as they have the ability to trans-
form a great variety of inorganic and organic pollutants into harmless minerals, which
can then be recycled back into the environment. They have the ability to oxidize many
organic chemicals synthesized industrially, as well as those produced naturally through
normal biological processes, and bacteria are employed in wastewater treatment plants
for this purpose. Some can convert waste organic materials into methane gas, which is a
useful form of energy. Others can transform inorganic materials, such as ammonium or
nitrate, which under certain circumstances may be harmful, into harmless forms, such
as nitrogen gas, the main constituent of air.
The functions of these microorganisms are not all beneficial to humans, however.
Some are the causative agents of disease and are responsible for many of the plagues
of the past, and, even today, some are responsible for major sickness and misery in the
world. Thus, the need is to protect humans from pathogens in water, food, and air,
while taking advantage of the prokaryotes' important abilities to cleanse pollutants
from waters bodies and soil and to recycle nutrients for sustainable production.

Morphology
The morphology of prokaryotes includes their shape, size, structure, and spatial
relationship to one another. Prokaryotes have three general shapes, as illustrated in
Figure 2.2. Those with spherical shape are cocci (singular, coccus), with a cylindrical
shape are rods or bacilli (singular, bacillus), and with a helical shape are spirilla (singular,
spirillum). Electron microphotographs of typical bacteria are illustrated in Figure 2.3.
(The photomicrographs in Figure 2.3, as well as in Figures 2.6, 2.7, and 2.8, come
from the Microbe Zoo, a project of the Center for Microbial Ecology at Michigan State
University, which permits their use in this text. The Microbe Zoo was created by Steven
Rozeveld, Joanne Whallon, and Cathy McGowan, and it is copyrighted by the Trustees
of the Michigan State University. Readers can find more images of microorganisms at
http:/ I commtechlab.msu.edu/ sites/ dk-me.)
Prokaryotes generally vary in width from 0.5 to 2 µrn and in length from 1 to 5 µm.
Cocci generally have a diameter within the 0.5- to 5-µm range as well. A gram of pro-
karyotes (dry weight) contains around 1012 prokaryotes. Because of the small size, the
surface area represented is about 12 m 2 /g. Thus, prokaryotes have a large exposure of
surface to the outside environment, permitting rapid diffusion of food into the cell and
very fast rates of growth.
Features of prokaryotic cells are illustrated in Figure 2.4, which also shows key
features of Eukarya. Although not all prokaryotes have identical structural features,
common to all are the external cell wall; the cytoplasmic membrane (also called plasma
membrane), which is just inside the cell wall; particles called ribosomes that are respon-
sible for protein production; the DNA, which is coiled in the nucleoid region; and an
internal fluid called the cytoplasm.
The cell wall in prokaryotes is a somewhat permeable rigid layer that is responsible
for keeping cells intact, as well as dictating cell morphology. Cell walls in Bacteria are
composed of repeating building blocks called peptidoglycan, which consists of two
sugar derivatives (N-acetylglucosamine and N-acetylmuramic acid) and a small group
of amino acids, such as alanine, glutamic acid, and either lysine or diaminopimelic
acid. In contrast, cell walls in Archaea are composed of a variety of materials, including
pseudomurein (also called pseudopeptidoglycan), which consists of two sugar deriva-
tives (N-acetylglucosamine and N-acetyltalosaminuronic acid) cross-linked with amino
 Bulcs of MlcrollloloCY 17

A B

c D

F11uH 2.3 Scanning electron microphotographs of ~pica! bacteria. (a) Staphytococcus

epldermldls. (b) Escherlchla coll. (c) Baell/us chains. {d) Leptospyra lnterrogans. (Source: With
permission from the Microbe Zoo and Bergey's Manual Trust, Michigan State University. Images a,
b, and c were created by Shirley Owens, and image d is from Bergey's Manual of Systematic
Bacteriology {Bergey and Holt, 2000).)

acids; thick units of repeating polysaccharides; and S-layers which consist of crystal-
line protein shells. The Archaeal cell wall renders them naturally resistant to certain
antibacterials, such as lysozyme (the anb'biotic in tears) and penicillin. Some unusual
Bacteria and An:haea do not possess cell walls, because either they live in osmotically
protected. habitafli, such as in animal cells, or they have unusually strong cell membrane
structures.
18 Chapter Twa

Nuclear region (chromosomal DNA) Pilus

Ribosome (RNA + protein)

Capsular material Cell membrane

A
Cytoplasm

Nuclear
membrane

Ribosomes

F1auRE 2.4 The typical structures of (a) prokaryotic cells, and (b) eukaryotic cells.

The cell membrane, or cytoplasmic membrane, is a phospholipid bilayer that lies

immediately beneath the cell wall and performs several important functions for the
cell. It is selectively permeable, is the main barrier to the passage of large molecules,
and controls the passage of nutrients into and out of the cell. It also is the location of
several important enzymes, including cytochromes, the enzymes that are involved in
electron transport and energy conservation. The cell membrane of most bacteria is quite
simple. It becomes more complex in autotrophic bacteria, which synthesize essentially
all cellular components from inorganic materials, and even more complex in the pho-
totrophic bacteria, which obtain energy from sunlight. In these latter organisms, the
cell membrane intrudes into the internal portion of the cell at certain points, providing
the additional membrane surface needed for the increased complexity and intensity of
functions.
Concentrated deposits of materials called cytoplasmic inclusions are contained in
some cells. Generally, they serve as storage for food or nutrients. Phosphate is stored as
 Basics af Mlcroblology 19

polymers in volutin granules, polysaccharide granules store carbohydrates, and other

granules contain polymerized ~-hydroxybutyric acid (PHH) or fatty materials. In cer-
tain sulfur-metabolizing bacteria, large amounts of sulfur accumulate in granules.
Cytoplasm is the fluidic material contained within the cell membrane that is used to
carry out cell growth and many cell functions. It consists of water, dissolved nutrients,
enzymes (proteins that enable the cell to carry out particular chemical reactions), other
proteins, and the nucleic acids---RNA and DNA. Also included are the densely packed
ribosomes, which are RNA-protein particles that carry out protein synthesis.
Important to all cells is the presence of DNA (deoxyribonucleic acid), a double-
stranded helix-shaped molecule that contains all the genetic information required for
cell reproduction and growth. It also contains in coded form all information required
to carry out the normal cell functions. This information is stored in the sequence of
nucleotides (each consisting of deoxyribose connected to one of four nitrogen bases--
adenine, guanine, cytosine, or thymine). The genetic information of the cell is stored,
replicated, and transcribed by the DNA molecule, which may also be called a prokary-
otic chromosome. Some bacteria also contain one or several much-smaller circular DNA
molecules, called plasmids, which also can impart additional phenotypic characteristics
to the organism.
The information contained in DNA is "read" and carried to the ribosomes for pro-
duction of proteins by RNA. RNA is single stranded, but otherwise similar to DNA.
The major difference is that the sugar portion of each nucleotide is ribose, rather than
deoxyribose, and uracil is substituted for the base thymine. RNA has three forms: mes-
senger RNA (mRNA), which carries the information for protein synthesis from the
DNA molecule; transfer RNA (tRNA), which carries amino acids to their proper site on
the mRNA for the production of a given protein; and ribosomal RNA (rRNA), which
functions as structural and catalytic components of the ribosomes, the sites where the
proteins are synthesized. DNA is the blueprint for the structure and functioning of the
bacteria, and the different forms of RNA can be viewed as the different workers that
read these plans and carry out the construction of the bacterial cell.
Some bacteria, such as those of the genera Bacillus and Clostridium, have the capabil-
ity of forming endospores (i.e., spores formed inside the cell). Spore formation generally
results when the environment approaches a state adverse to the growth of the bacte-
ria, such as the depletion of a required nutrient or unfavorable temperature or pH. In
the sporulated state, the microorganism is inactive. The spore has many layers over a
core wall, within which there are the cytoplasmic membrane, cytoplasm, and nucleoid.
Thus, it is similar to a vegetative cell except for the cell wall. It also contains about 10%
dry weight of a calcium-dipicolinic acid complex and only about 10% to 30% of the
water content of a vegetative cell. These characteristics help impart great resistance of
the cell to heat and chemical attack. An endospore can remain inactive for years, per-
haps centuries. When an endospore finds itself in a favorable environment for growth,
then it germinates back to the active vegetative state. Bacteria that form endospores are
quite difficult to destroy through normal sterilization techniques.
Certain external features of Bacteria or Archaea are useful for identification. Some
have a capsule or slime layer that is excreted from the cell proper and, because of its
viscous nature, does not readily diffuse away from the cell. This excreted material can
increase the viscosity of the surrounding fluid and can also help to hold bacteria together
in aggregates, such as bacterial "floes." Such layers are also significant in microorgan-
ism attachment to surfaces, such as in the formation of biofilms. Most capsule or slime
20 Chapter Twa

layers consist of polysaccharides of several types (glycocalyx) and polymers of amino

acids. Some slime layers are readily visible (capsules) and others are not (slime layers).
Removal of these external layers does not adversely affect the normal functioning of
the bacterial cell.
Other external features of some prokaryotes are flagella. These hair-like structures
are attached to the cytoplasmic membrane and protrude through the cell wall into
the surrounding medium. The length of the flagella may be several times that of the
cell. The number and location of the flagella are different for different species. Some
may contain a single terminal (polar) flagellum, some may have several at one end,
some may have several at both ends, and others may have them distributed over the
entire cell (peritrichous). Most species of bacilli have flagella, but they are rare in cocci.
Flagella are responsible for mobility in bacteria. When they rotate at high speed, they
can push or pull a microorganism a distance equal to several times its own length in
a second. Some bacteria, however, can move on surfaces without flagella by an action
called gliding, although this mode of motion is quite slow by comparison. Cells can be
caused to move toward or away from chemical or physical conditions, which is called
a tactic or taxes response. Movement in response to chemical agents is called chemotaxis,
and that in response to light is termed phototaxis.
Structural features that are similar to flagella are fimbriae and pili. Fimbriae are
more numerous than flagella, but considerably shorter and do not impart mobility.
They may aid microorganisms in attachment to surfaces. Pili are less numerous than
fimbriae, but are generally longer. They too aid in organism attachment to each other
and to surfaces. An example is the use of pili by E. coli for clumping together and
for attachment to intestinal linings. Pili appear to be involved in bacterial conjuga-
tion, an important process by which genetic information from one cell is transferred
to another. Conjugation appears to be how resistance to pesticides and antibiotics
and the ability to degrade toxic chemicals are transferred between microorganisms.
Pili also appear to be involved in extracellular electron transport, which enables
microbial cells to shuttle electrons among themselves or to solid surfaces, such as
electrodes.
Another characteristic property of prokaryotes is the way they are grouped together,
a feature that is related to the manner in which they reproduce. As discussed in detail
later, bacteria multiple by binary fission, or simply fission, which means one organism
divides into two. The two may remain attached together for some time, even after each
divides again. Because of varying tendencies among different species to cling together
after division and the differing ways in which they divide, characteristic groupings of
bacteria emerge. For this reason, bacteria may occur singly or in pairs (diplococci). They
may form short or long chains (streptococci or streptobacilli), may occur in irregular clus-
ters (staphylococci), may form tetrads (tetracocci), or may form cubical packets of eight
cells (sarcinae). These particular groupings can be observed with the optical microscope
and can aid in identification.

Chemical Composition
In order for microorganisms to grow and maintain themselves, they must have avail-
able essential nutrients, such as carbon, nitrogen, phosphorus, and sulfur for synthesis
of proteins, nucleic acids, and other structural parts of the cell. These requirements must
be met whether the bacteria are in nature, treating a wastewater, or detoxifying hazard-
ous contaminants. If these elements are not present in available forms in a wastewater
 Basics af Mlcroblology 21

to be treated, they must be provided as part of normal treatment plant operation. An

idea of the quantity of different nutrients needed to grow the cells can be obtained by
combining an estimate of the rate of cell production with the quantity of each element
in the cell.
Table 2.3 contains a summary of the general chemical characteristics of bacteria,
which contain 70% to 80% water. The water internal to the cells is extremely difficult to
remove by normal dewatering processes; thus, "dewatered" biological solids or sludge
still contain at least 75% water, an amount that is too large to enable incineration with-
out the addition of supplemental fuel.

TABLE 2.3 Chemical and Macromolecular Characteristics of Prokaryotic Cells

Chemical Compo8'tlon
ConstHuent Percentap
water 75
Dry Matter 25
Organic 90
c 45-55
0 22-28
H 5-7
N 8-13
Inorganic 10
P20s 50
~o 6.5
N~O 10
MgO 8.5
cao 10
so3 15
Macromolecular Composition E. coll and S. typhlmurlum"
Percentage~ Percentage Molecules per cell
Total 100 100 24,610,000
Proteins 50--60 55 2,350,000
Carbohydrates 10-15 7
Lipids 6-8 9.1 22,000,000
Nucleic Acids
DNA 3 3.1 2.1
RNA 15-20 20.5 255,500

•Data from Madigan, Martinko, and Parker (1997) and Neidhardt and Curtis (1996). E. coli dry weight
for actively growing cells is about 2.8 x 10-13 g.
b Dry weight.
22 Chapter Twa

Dried biosolids, the material remaining after all cellular water is evaporated by
heat drying at 105°C, contain about 90% organic matter, about half of which is the
element carbon, one-quarter is oxygen, and the remainder is divided mostly between
hydrogen and nitrogen. Nitrogen is an important element in proteins and nucleic
acids, which together represent about three-fourths of the total organic matter in
the cell. While air contains nearly 80% nitrogen, it is present in the zero-valent form
(N2), which is not available to most prokaryotes, except for a few with the ability
to fix atmospheric N 2 • Nitrogen fixation is a rather slow process and usually is not
adequate to satisfy nitrogen needs in engineered biological processes. When grown
in the presence of sufficient nitrogen, the bacterial cell will normally contain about
12% nitrogen by dry weight. The content can be reduced to about one-half of this
value by nitrogen starvation, which lowers the protein content, but also slows the
rate of growth. Under such conditions, the carbohydrate and lipid portions of the cell
increase.
Another necessary element for bacterial growth is phosphorus, which is an essen-
tial element in nucleic acids and certain key enzymes. Phosphorus may be added in
the form of orthophosphates to satisfy this need, the mass required generally being
one-fifth to one-seventh of that for nitrogen on a weight basis. As indicated in Table 2.3,
sulfur and iron also are required in significant amounts. Both are important in certain
enzymes.
Some elements are not required by all bacteria, but may be for some as critical com-
ponents of key enzymes, such as molybdenum for nitrogen fixation, nickel for anaero-
bic methane production, and cobalt for reductive dechlorination. Many microorganisms
require specific organic growth factors that they cannot produce themselves, such as
vitamins. Often, these are produced by other organisms living in mixed cultures, so that
they need not be added externally except when growing as a pure culture.
Empirical formulas for bacterial cells are useful when making mass balances for
biological processes. Empirical formulas based upon the relative masses of the five
major elements in cells are summarized in Chapter 5, with Cs1fp~ being the most
common formulation for design computations. The empirical molecular weight with
this formulation is 113 g, for which nitrogen represents 12.4% and carbon 53%, on a
weight basis.

Reptoduction and Growth

Knowledge of rates of bacterial growth and reproduction is essential for design of
engineered biological treatment systems, as well as for understanding the actions of
microorganisms in nature. Microorganisms generally reproduce through binary fission,
in which a cell divides into two after forming a transverse cell wall or septum. This
asexual reproduction occurs spontaneously after a growing cell reaches a certain size.
After reproduction, the parent cell no longer exists, and the two daughter cells normally
are exact replicates (clones) of each other, both containing the same genetic information
as the parent. The interval of time required for formation of two cells from one is the
generation time, which varies considerably depending upon the particular organism
and the environmental conditions. It may be as short as 30 min, as with E. coli, or many
days for other organisms where growth rates are restricted by less energy availability
or other environmental conditions. With unrestricted growth of E.coli, one organism is
converted to two organisms after 30 min, four after 1 h, eight after 111.i h, and 16 after
2 h. After 24 h and 48 divisions, the total population, if unrestricted in growth, would
 Basics af Mlcroblology 23

equal more than mu cells and would have a dry weight of about 50 kg. In principle, the
weight would increase from 10-10 g to that of a small human in a single day! In reality,
environmental and nutritional limitations of the culture limit the growth long before
such a mass increase occurs, but the potential for such rapid growth is inherent in the
microbial cell.
Some bacteria have less common methods of reproduction. Some species of the
genus Streptomyces produce many reproductive spores per organism, and each can give
rise to a new organism. Bacteria of the genus Nocardia can produce extensive filamen-
tous growth, which, if fragmented, can result in several filaments, each of which can
give rise to a new cell. Some bacteria can reproduce by budding, with the stalk that
develops from the parent separating and forming a new cell.

Energy- and Carbon-Source Classifications

An important characteristic of microorganisms is that they possess a very wide variety
of energy sources they can use for growth and maintenance. How a microorganism
gains energy is identified by its troph, which is from the Greek word for food or nutri-
tion. Microorganisms that use energy from light are termed phototrophs, while those
obtaining energy from chemical reactions are tenned chemotrophs. Chemotrophs that
use organic chemicals for energy are chemoorganotrophs, and those that use inorganic
chemicals for energy as chemolithotrophs.
Phototrophs, which generally use C02 as a source of cell carbon, are also classified
into two types based upon the chemical from which electrons are obtained to reduce
C02 to form cell organic matter. Those that use Hp, oxidizing it photochemically into
0 2 and electrons, are termed oxygenic phototrophs. In contrast, anoxygenic phototrophs
generally live only in the absence of 0 2 and extract electrons from reduced sulfur com-
pounds, such as H 2 S or S0 ; H 2; or organic compounds, such as succinate or butyrate.
When H Sis used, it is converted into H and S0 , somewhat akin to the photochemi-
cal convb-sion of water, with H being us~d as the electron source for synthesis and S0
being excreted as an oxidized product. Different chlorophylls are used in anoxygenic
photosynthesis, compared with oxygenic photosynthesis, to capture the energy from
the light and extract the electrons.
Other common terms used to describe microorganisms are related to the carbon
source used for cell synthesis. Autotrophs use inorganic carbon, such as COz1 for cell syn-
thesis, while heterotrophs use organic compounds for cell synthesis. Chemolithotrophic
bacteria commonly also are autotrophic, and chemoorganotrophs commonly are het-
erotrophic. For this reason, the different terms relating to the energy source and to the
carbon source for chemotrophs are often used interchangeably.

Environmental Conditions for Growth

In addition to the nutritional needs for energy and cell synthesis, microorganisms
require a proper physical and chemical environment for growth. Factors of importance
here are temperature, pH, 0 2 partial pressure, and osmotic pressure. The rate of all
chemical reactions is influenced by temperature, and since microbial growth involves
a series of chemical reactions, their rate of growth is also greatly influenced by tem-
perature. Over a limited range for each microbial species, growth rates increase with
temperature, and, in general, they roughly double for each 10°C rise. At temperatures
beyond the normal range for a given species, key enzymes are destroyed, and the
organism may not survive.
Another Random Scribd Document
with Unrelated Content
 CHAPTER XIII.
A CASH BOY'S TROUBLES.

The next day Mr. Little asked: "Did you take that suit to my tailor for
alterations, Scott?"
"Thank you, sir," said Scott, coloring, "but I think I will get along for
the present with the suit I am wearing."
"What does that mean?" demanded Ezra Little, quickly.
"I don't care to wear Loammi's clothes."
"Oh, you are proud, are you?" sneered Mr. Little.
"If it were necessary I would do so, but I think I am entitled to a
new suit."
"On what do you base your claim?"
"On the money which I handed you, Mr. Little," replied Scott.
"We will not discuss this question," said Ezra Little, coldly. "I have
already told you that this money will be needed to pay your
expenses."
Scott did not reply.
"Well, what have you to say to that?"
"Nothing, sir."
"You have no just cause of complaint. I have offered you a suit
which, when altered, would be almost as good as new. If you
change your mind about accepting it, you may let me know."
"Very well, sir."
On Thursday evening Scott made a call at Seth Lawton's boarding
house.
"I am glad to see you, Scott," said Mr. Lawton, cordially. "But you
look sober."
"I feel so, Cousin Seth."
"Why is that? Anything unpleasant happened?"
"I applied to Mr. Little for a new suit. He declined to buy me one, but
said I could have an old suit of Loammi's altered over for me."
"Didn't you mention the money you had placed in his hands?"
"Yes, but he said I was not earning my board, and this would make
up the deficit."
Seth Lawton rose from his chair and paced the room. It was his
habit to do so when he was disturbed.
"I didn't think Ezra Little would be so mean, though I knew he was
far from liberal. What did you say to his proposal?"
"I declined it. Loammi is not as large as I am, and, besides, I don't
feel like wearing his second-hand clothes when Mr. Little has money
of mine in his possession."
"What do you think of his claim that your services do not pay for
your board?"
"Judging from what I have found out about the pay of other
salesmen, I think that I earn more than my board."
"I think so, too. So you are to have no new suit?"
"No, sir."
"Perhaps you will be luckier than you imagine. You must remember
that I am your relative as well as Ezra Little. I will buy you a suit."
"But, Cousin Seth, I don't want to put you to that expense. You will
need all your money yourself."
Seth Lawton smiled.
"I will promise not to put myself to any inconvenience," he said.
"Will that satisfy you? Will you now refuse a favor at my hands,
Scott, my boy?"
"I would rather receive a favor from you than from Mr. Little, if you
really feel that you can afford it."
"You need not be apprehensive on that score. At what time do you
go out to lunch?"
"At twelve o'clock."
"I will call at that time to-morrow, and we will manage to get time to
stop at a tailor's and leave your measure."
"But, Cousin Seth, a ready-made suit will answer."
"As this is the first present I have given you, I will make it a good
one. Probably we can find a tailor near your store."
"Yes; Mr. Little's tailor has a shop only three blocks away. Here is his
card."
"The very thing."
When the suit was finished Scott put it on at once, and left his old
one to be cleaned and repaired.
It was hardly to be supposed that it would escape the observation of
Loammi and his father. As a matter of fact, it was handsomer than
any his cousin wore.
"Where did you get that suit?" asked Loammi, in amazement.
"It was a present," answered Scott.
"From whom?"
"Cousin Seth."
Loammi was not slow in carrying the news to his father.
"Pa," he said, "see the new suit Mr. Lawton has given Scott."
Mr. Little put on his glasses and closely examined his young relative.
"Did you ask Mr. Lawton to buy you a suit?" he asked, abruptly.
"No, sir. I did not wish him to go to such an expense."
"It must have cost at least twenty-five dollars."
"I think it cost twenty-eight."
"Seth is a fool. He is probably poor, and could not afford such an
extravagant outlay."
"He told me he could afford it, and I had to take his word."
"It is better than my best suit, pa," complained Loammi.
"You shall have as good a one when you need it. It is only three
weeks since I bought you a suit."
"Was it a ready-made suit?" asked Loammi of Scott.
"No; it was made to order by the tailor your father mentioned to
me."
"You will soon get it shabby wearing it every day."
"I don't intend to do so. I left my old suit to be cleaned and
repaired."
"Well, you are provided for, for the present, thanks to Seth Lawton's
folly. I don't wonder he is poor if that is the way he manages. Do
you know if he has got work yet?"
"He told me part of his time was occupied."
"I suppose he has got a little job to do at bookkeeping. Possibly it
will pay him twenty-five dollars. On the strength of that he has
bought you a suit at twenty-eight dollars. Seth always was a fool.
When he finds himself in need, it won't do him any good to apply to
me."
It was clear that Mr. Lawton had not raised himself in the estimation
of his rich relatives by his kindness to Scott.
Among the cash boys who worked in the store was a pleasant-faced
boy, named William Mead. He was two years younger than Scott, but
the latter had taken special notice of him, and without knowing
much of him, had come to feel an interest in him.
Usually Willie, as he was called, was bright and cheerful, but one day
he appeared with a sad countenance.
"What is the matter, Willie?" asked Scott, when the two boys went
out together at the noon hour.
Scott bought his lunch at a neighboring restaurant, but the cash boy
brought his with him from home.
"I don't like to annoy you with my troubles."
"But they won't annoy me. Please think of me as a friend."
"Then I will tell you. I have a brother three years older than I am,
who earns six dollars a week. He has been sick for two weeks, and
my mother misses his wages. You know I only get two dollars and a
half a week."
"That is very small."
"Some of the stores pay more, but Mr. Little never pays more than
that to a cash boy. Next week our rent comes due, and as we have a
strict landlord, I am afraid he will put us out when he finds mother is
not ready with the rent."
"I am sorry for you, Willie," said Scott, in a tone of sympathy. "Have
you no friend you can call upon for a loan?"
"Our friends are as poor as ourselves."
"When does your rent come due?"
"Next Saturday."
"I will think whether I can do anything for you, I will see you again
to-morrow."
"But you are poor yourself. Mr. Little's son was at the store one day,
and I overheard him telling one of the salesmen that you were a
poor relation."
"He is not likely to let me forget that. I am not sure that I can do
anything for you, Willie, but if I can I will."
"You have already done me good by speaking kindly to me."
"Come in to lunch with me, Willie. A cup of coffee will do you good."
That evening Scott had arranged to call on Mr. Lawton. He decided
to tell him of the young cash boy's troubles. Seth Lawton's face
showed his sympathy.
"It is really a hard case," he said. "We must see if we can't do
something for your friend."
"I hope you don't think I was hinting this to you, Cousin Seth."
"I don't, but still you won't object to my doing something for the
boy."
"Mr. Little says you are foolishly generous, and this is why you keep
poor."
"He will never make himself poor by his generosity. If you have the
boy's address we will call upon him."
 CHAPTER XIV.
A HELPING HAND.

The cash boy and his mother lived in a westside tenement house.
Just in front of the house, Scott met Willie Mead with a loaf of bread
which he was bringing home from a neighboring bakery. His eye
lighted up with pleasure when he saw Scott.
"Do you live here, Willie?" asked Scott.
"Yes, we live on the fourth floor."
"I have brought a gentleman with me who may be able to help your
mother. We will follow you upstairs."
"You may not like to climb so high, sir," said the cash boy, turning to
Mr. Lawton.
"I think I can stand it for once," rejoined Seth Lawton. "I am a little
more scant of breath than when I was a young man, but I am still
good for a climb."
Willie started ahead and the two visitors followed him.
"We will stop here on the landing till you have told your mother she
is to have visitors," said Seth, considerately.
The boy opened a door and entered a rear room. He reappeared in a
short time, and said: "Come in, please."
The room was neat, but the scanty and well-worn furniture showed
evidences of dire poverty.
Mrs. Mead, a woman of forty, though poorly dressed, had a look of
refinement, though her face was sad and anxious.
As she watched the entrance of the visitors her eyes seemed riveted
upon Seth Lawton. She took a step forward.
"Surely," she said, "I cannot be deceived. This is Seth Lawton."
"You know me?" said Seth, in amazement.
"Yes, and you ought to know me. We were born in the same village."
"Mary Grant!" ejaculated Seth, after a brief scrutiny.
"That was my name. Now I am Mary Mead. I married, but my
husband is dead. But sit down. It does me good to see an old
friend."
"It seems incredible," said Seth, as he took the proffered seat. "We
met last in England, and now again under strange and unexpected
circumstances." Seth Lawton seemed moved, but his tone was one
of satisfaction.
"Yes, Seth, much has happened since we parted."
"How long have you lived in America?"
"Ten years."
"And when did your husband die?"
"Three years since. He left me nothing but the children, and it has
been a sad and sorrowful time. We have lived, but there have been
times when we have been on the verge of starvation. And you, how
has it been with you?"
"I have no right to complain. I have lived comfortably. You know
Ezra Little?"
"Yes, it was at my request that he took Willie into his store. But the
two dollars and a half a week, which he pays him, seems very
small."
"I should think so. Didn't he know how poor you were?" asked Seth,
indignantly.
"Yes, but he said he could not favor one cash boy more than the
rest."
"Then he might have made you a present."
"I don't think it ever occurred to him, Seth. But how did you find
me? Did he give you my address?"
"No, that was not likely. Scott Walton—you must have known his
mother, my cousin Lucy—works in the same store. It was he who
heard of your trouble and reported it to me. Now tell me how you
are situated."
"We are likely to be turned out of these poor rooms, because we
cannot pay the rent. My eldest boy, Sam, has been sick, and as he
earned six dollars a week, it took most of our income from us. Next
week I think he will be able to go to work again."
"This is a poor place for you, Mary."
"We are glad of even this shelter. We are too poor to be particular."
"Your income consists only of what the two boys earn?"
"I earn something by sewing, but I have no sewing machine, and
the prices paid are very low. Still, every little helps."
"If you had a whole house and kept lodgers, you could make a
better income."
"No doubt, and I think I could do it if I had the means. But with no
capital, that is out of the question," she finished, with a sigh.
"I have a proposal to make to you. I have a room in a house on
West Sixteenth Street. It is a moderate sized house, and is to let
furnished. My present landlady is desirous of giving up the house, as
she wishes to be with her mother in the country, but she is tied by a
lease. Suppose you take it off her hands?"
"I should like nothing better, but you can judge whether an offer
from one so poor as myself would be accepted."
"Don't trouble yourself about that," said Seth Lawton, quietly. "I will
arrange it all, and will retain my room. I may say that the rooms are
all taken, so that you would be sure of an income at once."
"I should like the arrangement very much, and I should like
especially to have you with me, Seth; but it seems like a dream."
"We will make it a reality. I will see Mrs. Field this evening, and call
on you again to-morrow. When does your month here expire?"
"In three days."
"The time is short, but it is sufficient. You will hear from me very
soon. Meanwhile accept this small favor." He drew from his pocket a
ten-dollar note, and handed it to the widow.
"You are too kind, Seth," she said, gratefully. "You look poor
yourself, and——"
"I never was in the habit of dressing very handsomely," said Mr.
Lawton, smiling, "and just at present I look shabbier than usual.
Perhaps I have an object in it. At any rate, it is a fact. The help I
offer you will not embarrass me in the least."
"What a difference between you and Ezra Little," said Mrs. Mead.
"He has never offered me a dollar, though he knew me as well as
you."
"He acts according to his nature, Mary. Scott is an orphan—his father
died on the ship that brought them over from England—but Ezra
treats him as meanly as he has treated you and your boy. He makes
him work for his board, and has refused him a suit of clothes,
though he stood in need of it."
Mr. Lawton remained for half an hour. Then he rose, and went
downstairs, followed by Scott.
"It is strange you should have met an old acquaintance, Cousin
Seth," said Scott.
"More than an acquaintance, Scott. It may seem strange to you that
an old fellow like me should ever have been in love, but the time
was when I was in love with Mary Grant, and asked her to be my
wife."
"And she refused you?"
"Yes, Scott; I was fifteen years her senior, and she liked the man,
whom she soon after married, better. It was this disappointment
chiefly that led to my leaving England. I am very glad to have met
Mary again. Though years have passed I have not lost my
attachment for her. I am glad indeed that I can do the poor woman
a service."
His voice softened as he spoke, and it was clear that his early
romance was not dead.
"Mr. Mead was a handsome man," continued Seth. "You can judge of
that, for the boy Willie looks like him. He made a good husband, I
presume, but he had not the knack of succeeding in life."
"Like Mr. Little."
"Yes, like Ezra Little."
It occurred to Scott that the same thing might be said of Seth
Lawton himself, but he would not, of course, speak of it. He was
beginning to have a sincere respect and regard for Cousin Seth.
What matter if he were poor—at least compared with Ezra Little—he
evidently had a kind heart, and was inclined to be generous beyond
his means.
"All cannot become rich," said Scott. "I wish you had Mr. Little's
money, though."
"Don't wish that, Scott, for without that Ezra would be poor indeed.
It is all that he has to boast of."
"I am afraid it will be the same with Loammi."
"With this difference: Ezra, with all his faults, is enterprising and
industrious, and I don't think his son will be either. In the race of life
you may eclipse him, after all."
"It doesn't seem much like it now."
"No, but you are young yet, and time often works wonders."
"Won't it cost a good deal to set up Mrs. Mead in her new business?"
asked Scott, thoughtfully.
"Not very much. She will enter into a house fully furnished and
equipped, and with a sure and prompt income from a good set of
lodgers."
"I hope she will succeed."
"I think she will. If Ezra would pay you wages, in place of giving you
a home in his house, you might take a room there, too."
"I wish I could."
"Well, it may come about some time. But look, there is Loammi."
Yes, it was Loammi, sporting a light cane, and evidently on very
good terms with himself.
"Good-evening, Loammi," said Cousin Seth.
"Good-evening, Mr. Lawton," responded Loammi, patronizingly. "Are
you and Scott taking a walk?"
"Yes; and you?"
"Oh, I have been to call on a schoolmate. His father's awful rich."
"We, too, have been to make a call—on the mother of one of your
father's cash boys."
Loammi turned up his nose.
"You keep fashionable company," he said.
"We are not fashionable, like you, Loammi," said Scott, smiling.
"No, of course not," answered Loammi, in a matter-of-course tone.
"Well, ta, ta!"
"I wonder how that boy will turn out!" said Cousin Seth, thoughtfully.
 CHAPTER XV.
THE CASH BOY'S PROMOTION.

Cousin Seth arranged everything as he had planned, and Mrs.

Mead's landlord, when he called, learned to his surprise that his poor
tenant was intending to move.
"Have you found cheaper rooms?" he asked.
"No, but I am going to take a whole house."
The landlord looked astonished.
"Where?" he asked.
"On West Sixteenth Street."
"Yet you have always been pleading poverty, and only last month I
had to wait two days for the last dollar of the rent."
"That is true; but an old friend has found me out, and will give me a
helping hand."
Of course, no more was to be said.
The removal was soon made, for Mrs. Mead had little to move, and
with Seth Lawton's efficient help, the widow found herself in
possession of her new establishment, with everything running
smoothly.
"Now," said Mr. Lawton, "I must see if I can't do something for
Willie. How much does Ezra Little pay him?"
"Two dollars and a half a week."
"That is too little."
"I don't think Mr. Little will pay more."
"Let him ask."
"I am afraid in that case he will lose his place. The last time Willie
asked for a raise he was angry."
"Very well, if he loses his place I will find him another. Or, stay, I will
ask Ezra myself."
"That will be better."
So Seth called the next evening on his rich relative. He was not
received with open arms, for Mr. Little was under the impression that
he wanted to borrow money.
"I can't give you much time to-night, Seth," said the merchant. "I
have a business engagement. Have you found anything to do?"
"I think I can see my way clear to a place as confidential clerk and
bookkeeper in a small office downtown."
"How much salary?"
"Possibly fifteen dollars a week."
"You had better accept. You are extremely lucky at your age to get
such an office."
"You wouldn't be satisfied with it, Ezra," returned Seth, with a smile.
"I? You are dreaming. What, a well-known and long-established
merchant to think of such a salary! You must be insane."
"Yet you are within five years as old as I am, Ezra."
"What does that matter? I take it there is considerable difference
between your position and mine."
"Yes, I suppose so."
"To tell the truth, I didn't think you would be able to get any position
at all. I hope this won't slip through your fingers."
"Then you advise me to accept it?"
"Of course. You would be crazy not to do so. Remember, you will
have to depend upon yourself. The fact that you are a relation will
not justify you in asking help from me."
"I have a favor to ask, however, Ezra."
"I cannot lend you money, if that's what you mean," said Ezra,
brusquely.
"It isn't. I find that one of your cash boys is the son of an old friend
of ours—Mary Mead, formerly Mary Grant."
"Yes; I gave the boy a place in order to help her."
"You pay him two dollars and a half a week. There are only two
boys, and this is very small."
"It is all I pay any of the boys."
"But Willie is a well-grown boy of fourteen. Surely, out of old
friendship, and to help his mother, you can pay him more."
"Have you been talking to Mrs. Mead, and encouraged her to think
that I will increase her boy's wages?"
"Yes."
"Then you have done a foolish thing. I decline. I am half inclined to
discharge the boy."
"It won't be necessary. He will leave the store at the end of the
week."
"What does this mean?"
"That I will undertake to find him a better place."
Ezra looked annoyed and angry.
"You can't do it," he said. "You have no acquaintances in the city.
You are not even sure of employment yourself."
"So it seems you have sized me up, Ezra," said Seth Lawton, mildly.
"That is easy enough. You were born to be an unsuccessful man.
You are fifty-six years old, and I suppose you haven't saved enough
money to keep you going for three months."
"I don't owe a cent, Ezra."
"That is something. But I can't remain here talking. Don't forget
what I said about making sure of the place you spoke of."
"Just as I expected," thought Seth. "Ezra seems to be a thoroughly
selfish man. It is lucky for me that——" but he did not finish the
sentence.
Mr. Little did not think of the matter again till the superintendent told
him on Saturday night: "One of the cash boys has resigned his
place."
"Who is it?"
"William Mead."
"It is all the bad advice of Seth Lawton," he reflected. "He is a
perfect meddler. Probably his mother will be here in a day or two to
beg me to take him back."
But no such application came. Willie had obtained a place on Grand
Street at four dollars a week.
Scott continued to enjoy the companionship of Seth Lawton, but
sometimes Cousin Seth was out of the city for days at a time, in
which event Scott was thrown back on the company of Loammi, but
this gave him very little satisfaction.
One evening Loammi happened upon his cousin coming out of a
store on Sixth Avenue.
"Have you been buying anything?" he asked.
"Yes."
"What?"
"A couple of neckties."
"Where did you get the money?"
Scott said, quietly: "That is my business, Loammi."
"I thought you gave pa all the money you had."
"I gave him forty dollars."
"How much have you got left?"
"I don't care to tell."
This was enough for Loammi, who saw a chance to do his cousin an
ill turn. Accordingly he said to his father that evening: "Pa, did you
know that Scott had money?"
"What do you mean?"
Then Loammi told the story.
"I asked him how much he had, and he wouldn't tell me. It seems to
me he ought to have handed it to you."
In this Mr. Little agreed with his son.
"Call Scott," said he.
Scott was in his small chamber, and there Loammi found him.
"Pa wants to see you, Scott."
Scott went downstairs and into Mr. Little's presence.
"Do you wish to see me, sir?"
"Yes. Loammi tells me you have some money."
"Yes; I have a little money."
"I thought you gave up all you had when you came here."
"So I did, all but sixty cents, but I have regretted it since."
"Why?"
"Because I understood it was to be used for my clothing, and it was
not."
"I told you in what light I considered it. But I won't dwell upon that
now. You deceived me in letting me think you had given up all your
money."
"No, I did not, sir."
"Then how do you explain your having money at present. Was it
given you by Mr. Lawton?"
"No, sir."
"Where, then, did you get it?"
"It was money that I was swindled out of by a fellow passenger. I
induced him to return a part of it."
"How much have you now?"
"About five dollars."
"You may give it to me."
"I prefer not to do so, Mr. Little; I need it myself."
Scott spoke respectfully, but firmly.
"Do you refuse?" demanded Ezra, angrily.
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