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Lessons in Environmental
Microbiology
Lessons in Environmental
Microbiology

Authored by
Roger Tim Haug
CRC Press
Taylor & Francis Group
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© 2020 by Taylor & Francis Group, LLC

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Library of Congress Cataloging-in-Publication Data

Names: Haug, Roger Tim, author.

Title: Lessons in environmental microbiology / Roger Tim Haug.
Description: Boca Raton : Taylor & Francis, 2019. | Includes bibliographical references and index.
Identifiers: LCCN 2019003192 | ISBN 9781138336582 (hardback : alk. paper)
Subjects: LCSH: Microbial ecology.
Classification: LCC QR100 .H38 2019 | DDC 579/.17--dc23
LC record available at https://lccn.loc.gov/2019003192

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 To Peggy
My patient wife of 50 years
So looking forward to our next 50 years together
To our sons David and James
So proud to be your father

To Family
Contents

Preface.......................................................................................................................................... xxiii
Author........................................................................................................................................... xxv

1 Introduction to Environmental Microbiology...................................................................1

What Is Microbiology?.............................................................................................................1
Why Should I Study Microbiology?....................................................................................... 2
The Roster of Microbes............................................................................................................ 3
The Branches of Microbiology................................................................................................ 4
Environmental Microbiology.................................................................................................. 7

2 Oxidation and Reduction: The Energy Reactions of Life...............................................9

Introduction...............................................................................................................................9
Electrons and Life................................................................................................................9
Oxidation and Reduction – The Transfer of Electrons................................................. 10
The Atomic Nature of Things............................................................................................... 10
What Is an Electron?.......................................................................................................... 10
The Black Body Problem................................................................................................... 11
The Gold Leaf Experiment............................................................................................... 11
Absorption and Emission Spectra................................................................................... 12
The Double Slit Experiment............................................................................................. 12
The Periodic Table of Elements............................................................................................. 14
Electron Shells and Orbitals.................................................................................................. 16
Who Has the Electrons?......................................................................................................... 17
The Outermost Valence Electrons................................................................................... 17
Ionic and Pure Covalent Bonds........................................................................................ 18
Polar Covalent Bonds........................................................................................................ 19
Electronegativity................................................................................................................ 19
Oxidation States and Numbers........................................................................................ 20
Electron Donors and Acceptors............................................................................................ 23
The Common Electron Donors........................................................................................ 23
The Common Terminal Electron Acceptors................................................................... 23
Approaches to Balancing Redox Reactions......................................................................... 24
Balancing the Whole Reaction, the Eight Step Plan...................................................... 24
Balancing with Half-Cell Reactions................................................................................30
Electrons and COD/BOD....................................................................................................... 31
Summary.................................................................................................................................. 32

3 The Chemistry of Carbon (for the Non-Chemist in All of Us).................................... 35

Introduction............................................................................................................................. 35
Organic vs Inorganic.............................................................................................................. 35
Carbon, the Enchanted Element........................................................................................... 36
Water, the Wondrous Molecule............................................................................................. 38
Kekulé Structures................................................................................................................... 40

vii
viii Contents

Overview of Carbon Compounds........................................................................................42

Carbon Bonded with Hydrogen...........................................................................................43
Saturated with Hydrogen.................................................................................................43
Unsaturated with Hydrogen............................................................................................44
Conjugate Double Bonds – Life Gets Colorful...............................................................44
The Triple Bond.................................................................................................................. 46
Hydrocarbon Rings........................................................................................................... 46
Functional Groups with Oxygen.......................................................................................... 47
Alcohols............................................................................................................................... 47
Aldehydes and Ketones..................................................................................................... 49
Acids (Carboxylic Acids)................................................................................................... 50
Esters and Ethers................................................................................................................ 51
Combinations..................................................................................................................... 52
Stereoisomerism...................................................................................................................... 52
Functional Groups with Sulfur.............................................................................................54
Functional Groups with Nitrogen........................................................................................ 55
Amines................................................................................................................................ 55
Amides................................................................................................................................. 55
Amino Acids and the Peptide Bond................................................................................ 56
Nitro and Nitrate Compounds......................................................................................... 59
Functional Groups with Phosphorus................................................................................... 60
The Aromatics......................................................................................................................... 61
The Benzene Ring.............................................................................................................. 61
Substituted Benzenes........................................................................................................ 61
Polynuclear Aromatics......................................................................................................63
Heterocyclic Compounds......................................................................................................65
Natural Products and Foodstuffs......................................................................................... 68
Proteins................................................................................................................................ 68
Lipids, Waxes, and Membranes....................................................................................... 70
Fats and Oils.................................................................................................................. 70
Waxes and Paraffin....................................................................................................... 73
Phospholipids and the Membrane Bilayer................................................................ 73
Archaea and the Membrane Monolayer.................................................................... 74
The Steroids and Sterols............................................................................................... 75
Carbohydrates (the Sugars)............................................................................................... 76
Soaps and Detergents.............................................................................................................80
Synthetic Compounds............................................................................................................ 81
A Second Look at Hydrogen Bonding.................................................................................84
Summary.................................................................................................................................. 86

4 Life and Energy: The Principles of Chemical and Photo Thermodynamics............. 89

What Is Thermodynamics?................................................................................................... 89
The Laws of Thermodynamics.............................................................................................90
The Equivalence of Heat and Work.................................................................................90
Quantifying Heat Flow..................................................................................................... 92
The First Law...................................................................................................................... 93
The Concept of Enthalpy.................................................................................................. 95
Standard States and Standard Conditions..................................................................... 96
Determining the Enthalpy of Formation........................................................................ 97
Contents ix

Enthalpy Is a Property...................................................................................................... 99
Entropy and the Second Law......................................................................................... 101
Open and Closed Systems.............................................................................................. 103
The Third Law.................................................................................................................. 104
The Gibbs Free Energy......................................................................................................... 104
The Effect of Entropy....................................................................................................... 104
The Free Energy of Formation....................................................................................... 106
The Direction of Reaction............................................................................................... 106
Adjusting to Actual Concentrations.............................................................................. 109
Activity versus Concentration....................................................................................... 110
Gibbs Free Energy and the Equilibrium Constant...................................................... 113
The Effect of Temperature on Equilibrium.................................................................. 114
Coupled Reactions........................................................................................................... 114
The Equilibrium Constant for Dissolved Gases.......................................................... 114
Storing Gibbs Free Energy in Ion Gradients..................................................................... 117
The Thermodynamics of Light........................................................................................... 123
What Is Light?................................................................................................................... 123
The Electromagnetic Spectrum...................................................................................... 124
Enter Quantum Physics.................................................................................................. 126
Wave–Particle Duality and Spacetime.......................................................................... 129
A Definition of Light?...................................................................................................... 130
The Solar Constant........................................................................................................... 131
The Solar Spectrum......................................................................................................... 132
Photosynthetically Active Radiation (PAR)................................................................. 133
Photosynthetic Photon Flux Density (PPFD)............................................................... 133
The Laws of Microbes.......................................................................................................... 136
The First Law of Microbes.............................................................................................. 136
The Second Law of Microbes......................................................................................... 136
Summary................................................................................................................................ 137

5 Metabolic and Nutritional Classifications..................................................................... 139

Introduction........................................................................................................................... 139
Relationship to Oxygen........................................................................................................ 139
Aerobic Metabolism (Sometimes Called Oxic)............................................................ 139
Anoxic Metabolism (Sometimes Called Anaerobic Respiration).............................. 140
Anaerobic Metabolism (Also Called Fermentation)................................................... 143
Relationship to Environmental Factors............................................................................. 144
Temperature...................................................................................................................... 144
Hydrogen Ion and pH..................................................................................................... 147
Osmotic Pressure (Salinity)............................................................................................ 149
Barometric Pressure......................................................................................................... 150
Other Relationships......................................................................................................... 150
Nutritional Categories.......................................................................................................... 150
Energy, Electrons, and Carbon....................................................................................... 150
Chemolithoautotrophs.................................................................................................... 151
Chemoorganoheterotrophs............................................................................................ 155
Photolithoautotrophs....................................................................................................... 155
Photoorganoheterotrophs............................................................................................... 158
Other Minor Categories.................................................................................................. 159
x Contents

Chemolithoheterotrophs............................................................................................ 159
Photolithoheterotrophs............................................................................................... 159
Categories That Simply Don’t Exist............................................................................... 159
Case Studies........................................................................................................................... 160
The Extremophiles of Yellowstone and Rotorua......................................................... 160
Extreme Halophiles of the Great Salt Lake.................................................................. 164
The White Water Incident............................................................................................... 166
Summary................................................................................................................................ 168

6 The Synthesis Reactions of Microbial Life.................................................................... 171

Introduction........................................................................................................................... 171
Composition of a Microbial Cell......................................................................................... 171
Sources of Cell Carbon......................................................................................................... 172
Developing the Synthesis Reaction.................................................................................... 173
Using the Cell Formula C5H7O2N.................................................................................. 173
Using Organics for Cell Carbon..................................................................................... 174
Nitrogen Assimilation and Fixation.............................................................................. 176
Using CO2 for Cell Carbon.............................................................................................. 180
Thermodynamics of the Synthesis Reactions................................................................... 183
The Free Energy of Cell Formation............................................................................... 183
Free Energy Requirements Using Organics for Synthesis......................................... 185
Free Energy Requirements Using CO2 for Synthesis.................................................. 186
Summary................................................................................................................................ 189

7 Thermodynamics and Cell Yield...................................................................................... 191

Introduction........................................................................................................................... 191
The Cell Yield Coefficient, Y................................................................................................ 191
Cell Yield from Organic Redox Reactions......................................................................... 192
Organic Usage.................................................................................................................. 192
Energy Capture................................................................................................................ 193
Energy Required for Synthesis...................................................................................... 194
Energy Required for Nitrogen Reduction.................................................................... 194
Combined Equation......................................................................................................... 195
The Yield Coefficient....................................................................................................... 195
Calculation Procedures................................................................................................... 195
A Few Things to Note..................................................................................................... 199
Cell Yield from Inorganic Redox Reactions...................................................................... 200
Cell Yield Using Light Energy for Growth....................................................................... 205
Energy Captured from Photosynthesis........................................................................ 206
Energy Required for Synthesis...................................................................................... 207
Combined Equation......................................................................................................... 207
Photosynthetic Efficiency................................................................................................ 208
Photosynthetic Yield and Specific Growth Rate.......................................................... 209
Combining Energy and Synthesis Reactions.................................................................... 210
Problem Type 1: Using Calculated Y Values................................................................ 211
Problem Type 2: Using Assumed Y Values.................................................................. 212
Final Thoughts...................................................................................................................... 217
Summary................................................................................................................................ 217
Contents xi

8 Historic Moments in Microbiology and Public Health............................................... 219

The Microscope and the Kingdom Protista...................................................................... 219
The Binomial Naming System............................................................................................ 220
Spontaneous Generation and Vital Force.......................................................................... 221
Sterilization and Pasteurization......................................................................................... 224
Culturing Techniques...........................................................................................................225
The Germ Theory of Disease.............................................................................................. 227
Staining.................................................................................................................................. 228
Aseptic Surgical Practices.................................................................................................... 230
Immunology and Serology.................................................................................................. 231
Variolation and Smallpox............................................................................................... 231
Pasteur and Rabies........................................................................................................... 232
Serums for Diphtheria..................................................................................................... 232
The Story of Polio............................................................................................................. 233
Microbial Ecology (Soil Microbiology).............................................................................. 235
Chemotherapy (from Salvarsan to Sulfa to Penicillin).................................................... 236
Ehrlich and Salvarsan...................................................................................................... 236
Domagk and Sulfa........................................................................................................... 236
Penicillin and the Age of Antibiotics............................................................................ 238
Antibiotic Resistance....................................................................................................... 240
Probiotics................................................................................................................................ 241
The Discovery of Viruses..................................................................................................... 242
Disinfection Theory.............................................................................................................. 242
Molecular Biology................................................................................................................. 243
Epidemics and Pandemics................................................................................................... 244
London and Cholera, the Blue Death............................................................................ 245
Chicago and Typhoid Fever............................................................................................ 249
Los Angeles and Sewage Farming................................................................................ 251
The Great Sanitary Awakening.......................................................................................... 251
The Modern Era Begins....................................................................................................... 253

9 The World of Microbes (And a Few Related Friends).................................................. 257

Taxonomy and Phylogeny................................................................................................... 257
Prokaryotic and Eukaryotic Cell Types............................................................................. 257
Early Classification Schemes............................................................................................... 259
RNA Sequencing Leads to Domains and Beyond........................................................... 260
Prokaryotic Cell Structure................................................................................................... 262
Eukaryotic Cell Structure.................................................................................................... 264
The Bacteria........................................................................................................................... 265
The Eubacteria.................................................................................................................. 265
The Archaea...................................................................................................................... 267
Representative Phyla of Eubacteria............................................................................... 268
Proteobacteria (Mostly Gram-Negative Rods and Cocci)...................................... 268
Firmicutes (the Gram-Positive, Low G+C Bacteria)................................................ 271
Actinobacteria (the Gram-Positive, High G+C Bacteria)....................................... 272
Cyanobacteria.............................................................................................................. 273
Chlorobi and Chloroflexi........................................................................................... 274
Chlamydiae.................................................................................................................. 274
xii Contents

Spirochaetes................................................................................................................. 274
Bacteroidetes................................................................................................................ 274
Aquificae and Thermotogae...................................................................................... 275
Deinococcus-Thermus................................................................................................ 275
Phyla of the Archaea........................................................................................................ 275
Crenarchaeota.............................................................................................................. 275
Euryarchaeota.............................................................................................................. 276
Bacterial Strains................................................................................................................ 277
The Protista (the Protoctista)............................................................................................... 277
The “Animal-Like” Protozoa.......................................................................................... 278
The Photosynthetic Microalgae..................................................................................... 280
The Fungi............................................................................................................................... 282
The Molds.......................................................................................................................... 283
The Yeasts.......................................................................................................................... 287
The Fungi and Lignin...................................................................................................... 288
Some Important Metazoa.................................................................................................... 289
The Rotifers....................................................................................................................... 289
Tardigrades (Water Bears)............................................................................................... 289
Helminths......................................................................................................................... 291
Resistant Forms..................................................................................................................... 291
Acellular Infectious Agents (Akaryotic)............................................................................ 292
The Viruses....................................................................................................................... 292
Viroids............................................................................................................................... 294
Protein Infectious Particle (Prion)................................................................................. 294
Indicator Organisms............................................................................................................. 295
The Tree of Life..................................................................................................................... 296
Summary................................................................................................................................ 297

10 Infectious Diseases Important to Public Health and Sanitary Practice................... 301

Introduction........................................................................................................................... 301
Viral Diseases........................................................................................................................ 302
Poliomyelitis (Infantile Paralysis).................................................................................. 302
Hepatitis Type A (Infectious Hepatitis)........................................................................ 303
Influenza............................................................................................................................ 303
Gastroenteritis..................................................................................................................304
Smallpox............................................................................................................................304
Acquired Immune Deficiency Syndrome (AIDS)........................................................305
West Nile Fever.................................................................................................................305
Ebola (a Form of Hemorrhagic Fever)...........................................................................306
Bacterial Diseases.................................................................................................................306
Cholera...............................................................................................................................306
Salmonellosis.................................................................................................................... 307
Typhoid Fever................................................................................................................... 307
Dysentery..........................................................................................................................308
Escherichia coli O157:H7....................................................................................................308
Legionnaires’ Disease (Legionellosis)........................................................................... 310
Tuberculosis (TB), the White Death or Consumption................................................. 310
Contents xiii

Lyme Disease.................................................................................................................... 311

Anthrax.............................................................................................................................. 311
Trachoma........................................................................................................................... 311
Stomach Ulcers and Gastritis......................................................................................... 312
Bubonic Plague, the Black Death................................................................................... 312
Protozoan Diseases............................................................................................................... 313
Amoebic Dysentery or Amebiasis (Montezuma’s Revenge)...................................... 313
Giardiasis (Backpacker’s Diarrhea)............................................................................... 313
Cryptosporidiosis (Crypto)............................................................................................. 314
Malaria............................................................................................................................... 316
Trypanosomiasis.............................................................................................................. 316
Toxoplasmosis................................................................................................................... 317
Amoebic Brain Infection................................................................................................. 317
Fungal Diseases.................................................................................................................... 318
Aspergillosis..................................................................................................................... 318
Coccidioidomycosis (Valley Fever, San Joaquin Fever).............................................. 319
Farmer’s Lung................................................................................................................... 320
Candidiasis....................................................................................................................... 320
Histoplasmosis................................................................................................................. 320
Sporotrichosis................................................................................................................... 320
Helminth Diseases (Intestinal Worms and Flukes)......................................................... 320
Roundworms (Nematodes)............................................................................................. 321
The Flatworm Tapeworms (Cestodes).......................................................................... 325
The Flatworm Flukes (Trematodes).............................................................................. 326
Microbial Control by Heat Inactivation............................................................................. 327
Sterilization....................................................................................................................... 327
Pasteurization................................................................................................................... 328
Milk Pasteurization..................................................................................................... 328
Biosolids Pasteurization............................................................................................. 329
Bacterial Regrowth.......................................................................................................... 331
Summary................................................................................................................................ 332

11 Biochemistry and Bioenergetics (The Molecules of Life)........................................... 335

Introduction........................................................................................................................... 335
Tools of the Trade.................................................................................................................. 336
Building With Glucose......................................................................................................... 337
Using Glucose for Energy Storage................................................................................. 337
Using Glucose for Structure........................................................................................... 339
Using Glucose as a Tough Outer Hide.......................................................................... 341
Other Energy Storage Molecules........................................................................................ 341
Nucleotide Bases...................................................................................................................342
The Carriers of Energy and Electrons...............................................................................344
The ATP–ADP Energy System.......................................................................................344
The Electron Carrier Systems.........................................................................................346
Enzymes (Speeding Things Up).........................................................................................348
The Porphyrin Ring.............................................................................................................. 351
The Porphyrin Ring and Heme...................................................................................... 351
xiv Contents

The Porphyrin Ring and Chlorophyll........................................................................... 352

Peptidoglycan........................................................................................................................ 353
Oxygen and Nitrate as Electron Acceptors....................................................................... 356
Oxygen Free Radicals...................................................................................................... 356
Nitrate Reduction............................................................................................................. 357
Nitrogen Assimilation.......................................................................................................... 358
Assimilatory Nitrate Reduction..................................................................................... 358
Nitrogen Fixation............................................................................................................. 359
The Concept of the Common Intermediate...................................................................... 360
Biochemical Pathways in Heterotrophic Metabolism..................................................... 361
Glycolysis........................................................................................................................... 362
Fermentation Pathways................................................................................................... 363
The Kreb’s Cycle...............................................................................................................364
The Respiratory Chain and the Terminal Electron Acceptor.................................... 366
Chemiosmosis and the Proton Motive Force............................................................... 368
Other Metabolic Pathways.............................................................................................. 370
Hydrolysis.................................................................................................................... 370
General Oxidation....................................................................................................... 370
Beta Oxidation (β-Oxidation).................................................................................... 371
Protein Metabolism..................................................................................................... 372
Overall Metabolic Flow................................................................................................... 372
Metabolic Pathways of Photosynthesis.............................................................................. 373
The “Light” Reactions (Light-Driven Electron Flow)................................................. 373
The Bacterial One-Center System............................................................................. 373
The Two-Center or Tandem System......................................................................... 376
The “Dark” Reactions...................................................................................................... 378
The Calvin Cycle......................................................................................................... 378
The Problem of RuBisCo............................................................................................ 380
Carbon Concentrating Mechanisms......................................................................... 380
Combining the Light and Dark Reactions.................................................................... 381
Organization of the Light Absorbing Pigments.......................................................... 381
Energy Flow in the Cell....................................................................................................... 382
Information Macromolecules.............................................................................................. 383
Deoxyribonucleic Acid (DNA)....................................................................................... 383
Ribonucleic Acid (RNA).................................................................................................. 387
Messenger RNA (mRNA)........................................................................................... 387
Transfer RNA (tRNA) (Also Known as Soluble RNA or sRNA).......................... 387
Ribosomal RNA (rRNA)............................................................................................. 388
The Genetic Code............................................................................................................. 389
Final Overview...................................................................................................................... 391
Summary................................................................................................................................ 392

12 A Brief History of Life........................................................................................................ 395

Introduction........................................................................................................................... 395
What Is Life?.......................................................................................................................... 396
The Journey Starts with a Bang.......................................................................................... 396
Deep Time.............................................................................................................................. 398
Contents xv

The Earth Forms a Primordial Soup?................................................................................ 399

Prebiotic Chemistry on Earth......................................................................................... 399
Prebiotic Chemistry in the Cosmos............................................................................... 401
Was the Primordial Soup Enough?............................................................................... 402
Deep Sea Hydrothermal Vents – The Black Smokers?.................................................... 403
Deep Sea Hydrothermal Vents – The Lost City?..............................................................404
First Life.................................................................................................................................405
Stromatolites..................................................................................................................... 406
Banded Iron Formations.................................................................................................408
Ancient Graphite Layers................................................................................................. 410
Microfossils....................................................................................................................... 411
From Soup to the First Cell?................................................................................................ 411
Key Events in the Story of Life............................................................................................ 413
Is There Life Out There?......................................................................................................425
Seeking Intelligent Life................................................................................................... 426
Searching for Microbial Life........................................................................................... 427
Searching for Biomarkers............................................................................................... 427
Mirror Life on Earth?....................................................................................................... 428
Summary................................................................................................................................ 429

13 Kinetics and Biodegradability.......................................................................................... 431

Kinetics and Thermodynamics........................................................................................... 431
Catalysts and Enzymes........................................................................................................ 431
Mathematical Functions to Describe Reaction Rates...................................................... 433
Zero-Order Reactions...................................................................................................... 433
First-Order Reactions......................................................................................................434
Second-Order Reactions.................................................................................................. 435
Enzyme Kinetics................................................................................................................... 435
Single Enzyme–Substrate Model................................................................................... 436
Determining k and K M..................................................................................................... 439
Environmental Effects on Enzyme and Microbial Activity...........................................440
Effect of pH.......................................................................................................................440
Effect of Temperature......................................................................................................440
Enzyme Inhibition...........................................................................................................440
Microbial Reaction Rates..................................................................................................... 441
Effect of Temperature on Reaction Rates..........................................................................442
Substrate Biodegradability..................................................................................................443
A Question of Kinetics....................................................................................................443
Proteins, Lipids, and Sugars...........................................................................................444
Lignocellulosic Matter.....................................................................................................444
Respirometry.........................................................................................................................448
BOD and COD Testing....................................................................................................448
Constant Volume, Aerobic Respirometers.................................................................... 453
Constant Pressure, Aerobic Respirometers..................................................................454
Constant Pressure, Anaerobic Respirometers............................................................. 455
Case Study......................................................................................................................... 456
Complex Substrate Characteristics..................................................................................... 459
Summary................................................................................................................................ 461
xvi Contents

14 The Suspended Growth Bioreactor: Basic Concepts.................................................... 463

Types of Bioreactor Systems................................................................................................463
Classification of Liquid Phase Systems............................................................................. 463
Suspended Growth (Homogeneous Systems).............................................................. 463
Fixed Film (Attached Growth or Heterogeneous Systems).......................................464
Hybrid Systems................................................................................................................464
Solid Substrates (Particulate or Insoluble Substrates).................................................464
Growth and Substrate Utilization Equations...................................................................464
Growth Equation.............................................................................................................. 465
Substrate Utilization Equation....................................................................................... 465
Combined Growth Equation.......................................................................................... 466
The Four Kinetic Coefficients.............................................................................................. 467
Yield Coefficient............................................................................................................... 467
Maximum Substrate Utilization Coefficient................................................................ 468
Half-Velocity Constant.................................................................................................... 468
Endogenous Respiration Coefficient............................................................................. 468
The Batch, Suspended Growth Reactor............................................................................. 469
Substrate and Cell Mass Balances................................................................................. 469
The Log Growth Phase.................................................................................................... 471
The Log Death Phase....................................................................................................... 471
The Continuous, Stirred Tank Reactor (CSTR) without Recycle.................................... 472
Mass Balance on Cell Biomass....................................................................................... 472
Mass Balance on Substrate.............................................................................................. 474
Solids Retention Time...................................................................................................... 474
Minimum Solids Retention Time.................................................................................. 475
Bioreactor Response to SRT............................................................................................ 475
Minimum Effluent Substrate Concentration, Se min...................................................... 476
Design SRT........................................................................................................................ 477
COD of the Effluent Biomass.......................................................................................... 477
COD Mass Balance........................................................................................................... 477
Nutrient Requirements................................................................................................... 478
Maximum Cell Production Per Unit Volume............................................................... 479
Summary of Parameters.................................................................................................480
The CSTR with Effluent Recycle.........................................................................................483
CSTR with Biomass Separation and Recycle....................................................................484
Cell Mass Balance............................................................................................................ 485
Substrate Mass Balance................................................................................................... 486
Procedure for Solution..................................................................................................... 487
Practical Aspects.............................................................................................................. 487
Biomass Wastage.............................................................................................................. 488
Nutrient Requirements................................................................................................... 488
COD Balance..................................................................................................................... 488
Oxygen Requirements..................................................................................................... 489
Summary of Parameters................................................................................................. 489
CSTR with Biomass Recycle, Wastage from the Recycle Line, and Effluent
Biomass ≠ 0............................................................................................................................ 491
Calculation of SRT............................................................................................................ 492
Cell Mass Balance............................................................................................................ 492
Contents xvii

COD Balance..................................................................................................................... 493

Recycle Rate to Maintain Desired Xa............................................................................ 493
Summary of Parameters................................................................................................. 495
Summary................................................................................................................................ 495

15 The Suspended Growth Bioreactor: More Concepts and Some Variations............ 497
Modeling Multiple Rate-Limiting Substrates................................................................... 497
SRTmin under Bioreactor Conditions................................................................................... 498
Multiple Rate Limitations............................................................................................... 498
Aerobic SRT (aSRT)..........................................................................................................500
Combined Heterotrophic and Autotrophic Growth........................................................500
Food-to-Microorganism Ratio, F/M ................................................................................... 501
Chemostat Determination of Y, k, Ks, and b..................................................................... 501
Variations on the Basic Theme............................................................................................ 503
SBR (Sequencing Batch Reactor)....................................................................................504
MBR (Membrane Bioreactor)..........................................................................................504
IFAS (Integrated Fixed-Film Activated Sludge)...........................................................505
MBBR (Moving Bed BioReactor).................................................................................... 507
AGS (Aerobic Granular Sludge)..................................................................................... 507
Aerobic Digestion................................................................................................................. 509
Mass Balance.................................................................................................................... 510
Regulatory Requirements............................................................................................... 511
Self-Heating Variations................................................................................................... 511
Autothermal Thermophilic Aerobic Digestion (ATAD)........................................ 512
Dual Digestion............................................................................................................. 512
Advanced Simulation Models............................................................................................. 514
Summary................................................................................................................................ 514

16 The Suspended Growth Bioreactor: Operational Considerations............................ 517

Separation Means Success................................................................................................... 517
Flocculation and Granulation............................................................................................. 518
Mechanisms of Floc Formation...................................................................................... 518
Mechanisms for Granulation......................................................................................... 519
Measuring Biomass Settleability........................................................................................ 519
Characteristics of Biomass Settling............................................................................... 519
The Sludge Volume Index (SVI)..................................................................................... 521
Estimating the Return Sludge Concentration.............................................................. 523
Variations on the Standard SVI Test.............................................................................. 524
The Window of Operability................................................................................................ 524
Causes of Poor Settling........................................................................................................ 526
Non-Flocculated Growth................................................................................................ 526
Rising Sludge.................................................................................................................... 526
Filamentous Sludge Bulking.......................................................................................... 526
Highly Degradable Substrate.................................................................................... 527
Low N or P Concentrations....................................................................................... 527
Low or High pH.......................................................................................................... 527
Low Dissolved Oxygen (Low DO Bulking)............................................................ 528
High H2S Concentration (Reduced Sulfur Bulking).............................................. 528
xviii Contents

Low F/M Bulking........................................................................................................ 528

Viscous Sludge Bulking (Non-Filamentous Bulking)................................................. 528
Dispersed Growth and Pin Floc.................................................................................... 529
The Filamentous “Bad Boys”.............................................................................................. 529
Filament Identification.................................................................................................... 529
The More Common Filaments....................................................................................... 530
Quantifying Filament Abundance................................................................................ 535
Controlling Filamentous Forms.......................................................................................... 535
Process Operation............................................................................................................ 535
Chemotherapy to Stress the Filaments......................................................................... 536
Metabolic “de” Selectors................................................................................................. 536
Biological Foaming............................................................................................................... 538
Causes and Consequences.............................................................................................. 538
Controlling Foams........................................................................................................... 541
Biological Control Methods....................................................................................... 541
Physical/Chemical Control Methods....................................................................... 541
Summary................................................................................................................................542

17 Biological Nutrient Removal and Recovery...................................................................545

Introduction...........................................................................................................................545
The Problem of Eutrophication......................................................................................545
Nitrogen and Phosphorus as Growth Limiting Nutrients........................................546
Reasons for Nutrient Removal or Recovery................................................................. 547
The Search for Chemical Fertilizers..............................................................................548
Liebig’s Law of the Minimum........................................................................................ 550
Biological Nitrogen Removal.............................................................................................. 550
Nitrification Kinetics....................................................................................................... 551
Population Dynamics...................................................................................................... 552
SRTamin under Operating Conditions............................................................................ 553
Alkalinity Impacts........................................................................................................... 555
Alkalinity Loss from Nitrification............................................................................ 555
Alkalinity Gain from Denitrification....................................................................... 556
The Influence of pH......................................................................................................... 556
Processes for Nitrogen Removal.................................................................................... 558
The Three-Sludge System........................................................................................... 558
Two-Sludge Systems................................................................................................... 559
Single-Sludge Systems................................................................................................ 560
The Importance of the bCOD/TKN Ratio.................................................................... 565
Mass Balance for the MLE Process................................................................................ 567
Mass Balance for the Step-Feed Process....................................................................... 568
Supplemental Carbon Sources....................................................................................... 571
The Anammox Process................................................................................................... 571
Removal Capabilities....................................................................................................... 575
Biological Phosphorus Removal......................................................................................... 576
Normal Synthesis Uptake............................................................................................... 576
Enhanced Biological Phosphorus Removal (EBPR or Bio-P)..................................... 576
Historical Developments................................................................................................. 576
Mechanisms for EBPR..................................................................................................... 577
Contents xix

Processes for EBPR........................................................................................................... 580

Removal Capabilities....................................................................................................... 581
Sidestream EBPR.............................................................................................................. 582
Processes for Combined Nitrogen and Phosphorus Removal....................................... 583
Summary................................................................................................................................ 587

18 Anaerobic Processes for Methanogenesis...................................................................... 589

Introduction........................................................................................................................... 589
Applications........................................................................................................................... 590
Advantages and Disadvantages......................................................................................... 591
Bioreactor Types.................................................................................................................... 592
Suspended Growth.......................................................................................................... 593
Conventional Designs................................................................................................. 593
Anaerobic Contact....................................................................................................... 594
Covered Anaerobic Lagoons..................................................................................... 597
Packed Bed Anaerobic Processes................................................................................... 597
Anaerobic Filter........................................................................................................... 597
Fluidized Bed............................................................................................................... 597
The Upflow Anaerobic Sludge Blanket (UASB) Reactor............................................. 598
Plug-Flow Bioreactors...................................................................................................... 599
Operational Modes...............................................................................................................600
Single-Stage.......................................................................................................................600
Reactors in Series.............................................................................................................600
Temperature Phased Anaerobic Digestion (TPAD)....................................................600
Continuous Batch Thermophilic Anaerobic Digestion (CBTAD)............................. 601
Two-Phase (Acid/Gas Phase) Digestion....................................................................... 601
Dual Digestion.................................................................................................................. 602
Co-Digestion..................................................................................................................... 602
High Solids Digestion...................................................................................................... 602
Class A and B Biosolids....................................................................................................... 603
Process Microbiology...........................................................................................................604
Population Dynamics......................................................................................................604
Syntrophic Cooperation.................................................................................................. 608
Minimum Solids Retention Times................................................................................. 609
Design Solids Retention Times...................................................................................... 611
Biogas Characteristics.......................................................................................................... 611
COD Removal and Methane Equivalency.................................................................... 611
Biogas Composition......................................................................................................... 612
Biogas Quantity and Fuel Value.................................................................................... 613
Environmental Factors......................................................................................................... 615
pH and Alkalinity............................................................................................................ 616
The Natural Source of Alkalinity.................................................................................. 617
Adding Alkalinity........................................................................................................... 618
Nutrients............................................................................................................................ 619
Inhibition and Toxicity.................................................................................................... 620
Cations and Heavy Metals......................................................................................... 620
Sulfide Toxicity............................................................................................................ 621
Ammonia Toxicity....................................................................................................... 622
xx Contents

Monitoring and Testing....................................................................................................... 622

pH and Its Measurement................................................................................................ 622
Total Volatile Acids.......................................................................................................... 623
Bicarbonate Alkalinity.................................................................................................... 623
Other Control Tests.......................................................................................................... 624
Changing the Temperature Regime................................................................................... 624
Improving Biodegradability................................................................................................ 625
Enhanced Anaerobic Digestion..................................................................................... 626
Thermal Pretreatment/Thermal Hydrolysis............................................................... 627
Bioreactor Modeling............................................................................................................. 628
Final Thoughts...................................................................................................................... 632
Summary................................................................................................................................ 633

19 Principles of Biological Composting............................................................................... 635

What Is Composting?........................................................................................................... 635
Common Feedstocks............................................................................................................ 636
Historical Developments..................................................................................................... 636
Generalized Composting Schematic.................................................................................. 638
Feed Conditioning........................................................................................................... 639
Process Stages...................................................................................................................640
Compost as a Product...................................................................................................... 641
Biological and Process Fundamentals...............................................................................642
The Microbes of Interest.................................................................................................642
The Effect of Temperature..............................................................................................643
Moisture Content.............................................................................................................643
Free Air Space...................................................................................................................644
Oxygen Content................................................................................................................644
The Energy Balance.........................................................................................................644
Composting Systems............................................................................................................645
Nonreactor Processes......................................................................................................645
Reactor Processes.............................................................................................................650
Vertical Flow Processes..............................................................................................650
Horizontal and Inclined Flow Processes................................................................. 651
Nonflow, Static Bed (Batch) Processes...................................................................... 653
Completing the System...................................................................................................654
Pathogen Control..................................................................................................................654
Example Mass and Energy Balance................................................................................... 655
Summary................................................................................................................................ 657

20 Microbially Induced Corrosion........................................................................................ 659

Microbes vs Concrete and Mortar...................................................................................... 659
A Tale of Two Microbes........................................................................................................664
Solutions................................................................................................................................. 666
Summary................................................................................................................................ 669

21 Biological Air Pollution Control....................................................................................... 671

Introduction........................................................................................................................... 671
Compounds of Concern.................................................................................................. 671
Contents xxi

Odor Measurement.......................................................................................................... 672

Activated Sludge Diffusion................................................................................................. 673
The Biotrickling Filter.......................................................................................................... 674
The Early Years................................................................................................................. 674
To Lava Rock and Beyond............................................................................................... 675
Other Media Types.......................................................................................................... 677
Biofiltration............................................................................................................................ 679
System Description.......................................................................................................... 679
Filter Media.......................................................................................................................680
Loading Capacity.............................................................................................................680
Moisture Balance.............................................................................................................. 681
Temperature...................................................................................................................... 681
Acidity............................................................................................................................... 682
Hybrid Media...................................................................................................................684
Biogas Treatment...................................................................................................................684
Early Work.........................................................................................................................684
Explosion Limits............................................................................................................... 685
Development..................................................................................................................... 685
Summary................................................................................................................................ 688

22 Microbial Ecology................................................................................................................ 691

Introduction........................................................................................................................... 691
Acid Mine Drainage (AMD)................................................................................................ 692
Origin of the Problem...................................................................................................... 692
Acid Mine Drainage in South Africa............................................................................ 694
The Animas River Incident............................................................................................. 695
The Human Microbiome..................................................................................................... 697
What Is a Microbiome?.................................................................................................... 697
The Intestine as a Bioreactor........................................................................................... 698
The Intestinal Community............................................................................................. 699
The Importance of Microbial Diversity and Stability................................................. 700
Fecal Elimination............................................................................................................. 700
Cryptobiotic Soil Crust........................................................................................................ 700
The Winogradsky Column.................................................................................................. 704
The Theory........................................................................................................................ 704
The Practice....................................................................................................................... 706
The Grand Biogeochemical Cycles..................................................................................... 707
The Cycles of Carbon....................................................................................................... 707
The Reservoirs of Carbon........................................................................................... 707
The Slow Carbon Cycle.............................................................................................. 708
The Fast Carbon Cycle................................................................................................ 709
The Keeling Curve...................................................................................................... 710
Ocean Acidification..................................................................................................... 711
The Cycle of Nitrogen...................................................................................................... 713
The Cycle of Phosphorus, Life’s Bottleneck................................................................. 714
Harmful Algal Blooms (HABs)........................................................................................... 717
Occurrence........................................................................................................................ 717
Cyanotoxins...................................................................................................................... 720
xxii Contents

Microcystis aeruginosa....................................................................................................... 722

The Toledo Incident......................................................................................................... 723
Methane in the Environment.............................................................................................. 724
Anaerobic Oxidation of Methane.................................................................................. 725
Aerobic Oxidation of Methane....................................................................................... 725
The Deepwater Horizon Incident.................................................................................. 726
Summary................................................................................................................................ 727
References.................................................................................................................................... 729
Index.............................................................................................................................................. 737
Preface

I taught my first course in environmental microbiology in 1972, fresh out of graduate school
and a young professor in the civil engineering department at then Loyola University of
Los Angeles. You could count on a number of things back then. With rare exception, the
graduate students were engineers, and most had never taken a college course in biology,
let alone microbiology. The environmental movement had just exploded on the scene with
the first Earth Day held in 1970. There was a great deal of excitement over this new field
called sanitary or environmental engineering. More than a few students were aerospace
engineers hoping to retrain pending an expected slowdown after the Apollo moon land-
ings. Virtually every subject within the realm of environmental microbiology was new to
them. If they had heard of the DNA molecule, they didn’t know much about it or what the
letters stood for. No one had a calculator. We used slide rules. Things were simple.
But the situation changed dramatically in the almost half-century since that first class.
Science and technology moved forward at a dizzying pace. The environmental field
learned how to use nature’s microbes in new and clever ways. And the students changed
as well. Most now come into our graduate program much better prepared in the basic sci-
ences. Today’s class is usually about one-third engineers, one-third environmental science
majors, and the balance from various science backgrounds. While this diversity is wel-
come, it is a challenge at times to accommodate the varied backgrounds.
In my first years teaching environmental microbiology I used a course text Microbiology
for Sanitary Engineers by Professor Ross McKinney, Department of Civil Engineering,
University of Kansas. Many similar environmental engineering programs used this text.
It was 300 pages long, about the right size for a one-semester course, and focused on the
topics of most interest to students who were going on to become practitioners in the field.
But the book was published in 1962 and was showing its age. Everyone expected a second
edition but it never came. I tried many other textbooks in the subsequent years but was
never very happy with them. Microbiology textbooks intended for undergraduate biology
students are very big, very expensive, and usually focused on science and medical aspects.
I began to rely on my own notes, and over time these evolved into the book that you’re
holding.
One of my mentors once told me that most professors write books for other professors.
What he meant was that books were often written to impress rather than to teach. I have
found this often to be true. I once considered adopting a new book by one of my former
professors. It was a great book as long as you already knew the subject. A student trying to
learn the subject for the first time would be overwhelmed and probably give up.
My book was written to be a teaching tool. The audience is students who do not already
know the subject but want to learn and want to become practitioners in the field. Every
effort has been made to bring complex subjects down to Earth and present them in a way
that is both enlightening and entertaining. To accommodate the diverse backgrounds of
my expected readers, I have included chapters on subjects such as organic chemistry and
biochemistry. If you have a background in these subjects, you may wish to skip them
or give them a quick read. Remember, it is important for a practitioner to understand
the basic concepts of a subject like organic chemistry, but there is no need to become an
organic chemist. People in this field do just fine with a basic understanding of such sub-
jects. Also, example problems and solutions are presented throughout the book to help the

xxiii
xxiv Preface

aspiring student. In my experience, a student who can solve real problems usually has a
good command of the subject. He or she usually just needs some guidance. Example prob-
lems are a good way to provide it.
I have always found it fascinating to work with nature’s microbes to accomplish useful
and varied purposes. Over the years I have had the fortune of working in positions that
kept me actively involved with the practice of biological waste treatment. I have tried to
blend both theory and practice into this work. Through this book, I hope to share my fas-
cination with this subject and inspire you to become a life-long student of environmental
microbiology. It touches us all in so many ways throughout our lives.
Author

Dr. Roger Tim Haug obtained a Bachelor of Science in Civil

Engineering from Loyola Marymount University and then earned
a Master of Science and a Doctor of Philosophy in Environmental
Engineering from Stanford University. He has been a Registered
Professional Engineer in the State of California since 1974. He is a
member of the Water Environmental Federation, serving on the
Biosolids Advisory Board and the Bioenergy Sub-Committee; a
member of the California Water Environment Association; and a
life member of the American Society of Civil Engineers. In 2008,
Dr. Haug received the Gordon Maskew Fair Award, the high-
est honor of the American Academy of Environmental Engineers. Also in 2008, he was
awarded the Rufus Chaney Award from the U.S. Composting Council for research excel-
lence and outstanding service to the composting industry. Dr. Haug has authored over
120 publications on the technical and management aspects of wastewater collection and
treatment. Dr. Haug joined the faculty at Loyola Marymount University in 1971 in the
Department of Civil and Environmental Science. Beginning in 1975 he became a full-time
consulting engineer and served many clients in the areas of wastewater treatment, bio-
solids management, and composting. He joined the Bureau of Engineering, City of Los
Angeles, in 1990 and was charged with managing the capital improvement program for
the City’s wastewater treatment plants. In 1999, he was promoted to Deputy City Engineer
and Wastewater Program Manager with authority over the wastewater capital improve-
ment program. Dr. Haug retired from City service in 2010 and is now Emeritus Professor
of Civil and Environmental Engineering at Loyola Marymount University. He serves on
advisory committees for several public agencies and continues to teach his favorite course,
Environmental Microbiology.

xxv
1
Introduction to Environmental Microbiology

The importance of microorganisms can’t be overemphasized…it is estimated that microbes

contain 50% of the biological carbon and 90% of the biological nitrogen on Earth…they
greatly exceed every other group of organisms on the planet.

Willey et al. (2008)

Microbiologists like to say, ‘Microbes rule the Earth, and we just live on it.’

Gregory Fournier (2014)

What Is Microbiology?
Microbiology is generally defined as the study of living organisms too small to be seen
with the naked eye, the world of microorganisms or microbes. Our eyes can detect an
object down to about 0.2 mm, or 200 μ. Below that, individual specks are not visible to us.
Having been raised in our modern world, this may seem a somewhat strange division.
Why would it make any difference whether we can see an organism with our eyes or not?
We grow up immersed in the knowledge of an enormous universe, ranging from the very
small as seen through microscopes to the very large as seen through telescopes. We can
see down to the molecular level with electron microscopes and peer to the edge of time
with the Hubble Space Telescope. We grow up with the wonderful knowledge that instru-
ments can extend the range of our eyes and that the world includes far more than we can
see without them.
So why would we make a distinction between biology of the small (microbiology) and
biology of everything else (macrobiology)? The answer lies mainly in the history of the
science. When we study any science we cannot divorce ourselves from the history of that
science. Macrobiology, the study of living systems that we can see with our eyes, is as old
as mankind itself. The earliest humans had to learn about nature, both plants and animals,
to survive. Macrobiology was self-evident from the earliest time that man could contem-
plate his own being. Man’s attempts to organize and categorize the living world date to
ancient times.
But the world of microbes remained beyond the human senses, too small to be seen. Despite
this limitation, mankind learned to benefit from some of the reactions that microbes offer.
The fermentation of fruits to wine and the leavening of bread were known to the ancients.
But things began to change around the year 1600. Galileo pointed his crude telescope to
the sky, forever changing mankind’s view of his place in the universe. If lenses could make
distant objects seem closer, could lenses make small things appear bigger? Ironically, it was
almost a century after Galileo, about the year 1700, when a Dutch businessman, Anton van

1
2 Lessons in Environmental Microbiology

Leeuwenhoek (1632–1723), became an expert in grinding lenses and building microscopes.

Anton built the best microscopes of the day and discovered the previously unknown world
of microorganisms. His best scopes were believed to magnify almost 400 times, quite an
accomplishment when you consider that today’s best light microscopes magnify to about
1,000 times. He called the small beings “animalcules” and wrote many letters on his find-
ings to the Royal Society of London, the premiere scientific organization of the time.
But the study of microbes remained largely a curiosity. No one associated such small
“animalcules” with disease. It would take another 150 years for man to prove the associa-
tion of microbes with disease. While van Leeuwenhoek is considered by most as the father
of microbiology, the science of microbiology was born in the second half of the 19th cen-
tury. Because it developed so recently and because some of the microbes were identified as
causative agents of the great infectious diseases of that era, microbiology was viewed as its
own science. New tools and techniques had to be developed to study the microbes. People
began to refer to themselves as microbiologists to distinguish their trade from those that
studied the macro world: the biologists, zoologists, plant physiologists, paleontologists,
and numerous others. The techniques of this new science also distinguished its practitio-
ners from others. Sterilization and the use of culture media became the tools of their trade.
Today, we know that all life is a continuum and shares the same basic biochemistry.
Size doesn’t matter in that regard. In fact, common biochemistry has become one of the
grand unifying principles of all biology, whether micro or macro. Nevertheless, the study
of microbes deserves special attention for environmental engineers and scientists.

Why Should I Study Microbiology?

Microbes are largely responsible for the global cycles of carbon, nitrogen, and sulfur on
planet Earth. They decompose the organic materials produced by photosynthesis and,
thereby, save us from being buried in organic debris. They make some of our most cher-
ished products, such as wine, beer, cheese, yogurt, and sauerkraut. Microbes are already
important players in the growing biofuel industry. They manufacture fuel ethanol and
may someday be major sources of renewable biodiesel. Most recently, man has learned to
insert genes into these little biochemical factories to produce valuable molecules such as
human insulin and human growth hormone.
Microbes are the heart and soul of modern wastewater treatment, saving us from our
own effluents. In the 1970s, there was an effort to develop physical/chemical processes
with the idea of replacing biological processes. Having landed man on the moon in 1969,
many saw “space age” processes as the way forward. Several sewage treatment plants were
built based on physical and chemical processes. None survived the test of time. They were
complicated, expensive to operate, and could not match the effluent quality and reliability
of their biological counterparts. Since that time, the wastewater industry has learned to
adapt nature’s microbes in more and clever ways. Without doubt, biological treatment is
the “flagship” process for today’s wastewater treatment and water reclamation industries.
Ever wonder how many cells make up your body? If you’re like me, you’re naturally
thinking of your own human cells. It turns out that this number is not that easy to deter-
mine, but estimates range from about 5 to 50 trillion (1 trillion = 1012). It takes a lot of cells
to make you who you are. But there is general agreement that the vast number of human
cells is at least equaled by the number of bacterial cells in and on your body. Estimates on
Another random document with
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27.(a) Rind and fistulous appendages Oceanapia
present; microscleres sigmata
(b) No rind; skeleton reticulate; Gellius
microscleres sigmata and/or
toxa
28.(a) Skeleton confused or formed of 29
bundles of spicules with
echinating spined styles
(b) Skeleton fibrous or reticulate, or 45
formed of short columns
(c) Skeleton formed of a dense 52
central axis, and columns
radiating from it to the surface
29.(a) Spicules of the ectosome styles Pytheas
(b) Spicules of the ectosome oxeas Clathrissa
or absent
(c) Main skeleton confused. Spanioplon
Special ectosomal skeleton
absent
30.(a) Megascleres of the 31
choanosome not differing from
those of the ectosome
(b) Megascleres of the 32
choanosome differing from
those of the ectosome
31.(a) Chelae absent. 33
(b) Chelae present 44
32.(a) Trichodragmata present Tedania
(b) Trichodragmata absent 42
Fig. 110.—Megascleres. a-l and q-s, Modifications of monaxon type. a,
Strongyle; b, tylote; c, oxea; d, tylotoxea; e, tylostyle; f, style; g, spined
tylostyle; h, sagittal triod (a triaxon form derived from monaxon); j, oxytylote;
k, anatriaene; l, protriaene; m, sterraster (polyaxon); n, radial section
through the outer part of m, showing two actines soldered together by
intervening silica; o, desma of an Anomocladine Lithistid (polyaxon); q,
crepidial strongyle, basis of rhabdocrepid Lithistid desma; r, young form of
rhabdocrepid desma, showing crepidial strongyle coated with successive
layers of silica; s, rhabdocrepid desma.

33.(a) Skeleton reticulate or fibrous 34

(b) Skeleton radiate or diffuse 37
(c) Skeleton with radiating fibres forming a Quasillina
reticulum with others crossing them at
right angles
34.(a) No microscleres 35
(b) Microscleres sigmata and/or toxa with Desmacella
or without trichodragmata
35.(a) Sponge fan- or funnel-shaped 36
(b) Sponge not fan- or funnel-shaped Hymeniacidon
36.(a) Megascleres slender and twisted Phakellia
(b) Megascleres somewhat stout, not Tragosia
twisted
37.(a) Sigmata present, skeleton diffuse Biemma
(b) Sigmata absent 38
38.(a) Skeleton more or less radiate 39
(b) Skeleton diffuse; sponge boring Cliona
39.(a) Sponge discoid with marginal fringe Halicnemia
(b) Sponge massive or stipitate without 40
marginal fringe
40.(a) Sponge body prolonged into Polymastia
mammiform projections
(b) Sponge body without mammiform 41
projections
41.(a) No microscleres. Megascleres Suberites
tylostyles with or without styles
(b) Microscleres centrotylote. Megascleres Ficulina
styles or tylostyles
42.(a) Choanosomal megascleres smooth 43
(b) Choanosomal megascleres spined Dendoryx
43.(a) Microscleres chelae and sigmata of Lissodendoryx
about the same size
(b) Chelae, if present, smaller than the Yvesia
sigmata
44.(a) Isochelae Esperiopsis
(b) Anisochelae Esperella
45.(a) Fibres or columns plumose 46
(b) Fibres or columns ectyonine 47
46.(a) Microscleres toxa Ophlitaspongia
(b) Microscleres absent Axinella
47.(a) Skeleton reticulate 48
(b) Skeleton not reticulate 49
48.(a) Microscleres present. Spicules of the Myxilla
fibre core spined
(b) Microscleres absent. Spicules of the Lissomyxilla
fibre core smooth
49.(a) Main skeleton formed of plume-like 50
columns
(b) Main skeleton formed of horny fibres Clathria
(ectyonine). Special dermal skeleton
 wanting
50.(a) Dermal skeleton contains styles only Microciona
(b) Dermal skeleton contains diactine 51
spicules with or without styli
51.(a) Main skeleton columns with a core of Plumohalichondria
smooth oxeas
(b) Main skeleton columns with a core of Stylostichon
spined styles
52.(a) Central axis contains much spongin. Raspailia
Echinating spined styli present
(b) Central axis with little or no spongin. Ciocalypta
Spined styles absent. Pillars radiating
from the axis support dermal skeleton
53.(a) Ground substance between chambers Spongelia
clear; chambers pear-shaped or oval;
eurypylous
(b) Ground substance granular. Chambers 54
spherical with aphodi
54.(a) Fibres not pithed; sponge fan-shaped Leiosella
(b) Fibres pithed; sponge massive Aplysina
55.(a) Chambers long, tubular, branched Halisarca
(b) Chambers not much longer than Oscarella
broad; not branched
56.(a) Amphidiscs present Ephydatia
(b) Amphidiscs absent Spongilla

CHAPTER IX

PORIFERA (CONTINUED): REPRODUCTION, SEXUAL AND ASEXUAL—

PHYSIOLOGY—DISTRIBUTION—FLINTS
The reproductive processes of Sponges are of such great
importance in leading us to a true conception of the nature of a
sponge that we propose to treat them here in a special section. Both
sexual and asexual methods are common; the multiplication of
oscula we do not regard as an act of reproduction (p. 174).

Fig. 111.—A, amphiblastula larva of Sycon raphanus; B, later stage, showing

invagination of the flagellated cells. c.s, Segmentation cavity; ec, ectoderm;
en, endoderm. (After F. E. Schulze, from Balfour.)

A cursory glance at a collection of sponge larvae from different

groups would suggest the conclusion that they are divisible into two
wholly distinct types. One of these is the amphiblastula, and the
other the parenchymula. This was the conclusion accepted by
zoologists not long ago. We are indebted to Delage, Maas, and
Minchin for dispelling it, and showing that these types are but the
extreme terms of a continuous series of forms which have all the
same essential constitution and undergo the same metamorphosis.

The amphiblastula of Sycon raphanus (Fig. 111) consists of an

anterior half, formed of slender flagellated cells, and a posterior half,
of which the cells are large, non-flagellate, and rounded. These two
kinds of cell are arranged around a small internal cavity which is
largely filled up with amoebocytes. The flagellated cells are
invaginated into the dome of rounded cells during metamorphosis, in
fact, become the choanocytes or gastral cells; the rounded cells, on
the other hand, become the dermal cells—an astonishing fact to any
one acquainted only with Metazoan larvae.
A typical parenchymula is that of Clathrina blanca (Fig. 112). When
hatched it consists of a wall surrounding a large central cavity and
built up of flagellated cells interrupted at the hinder pole by two cells
(p.g.c)—the mother-cells of archaeocytes. Before the
metamorphosis, certain of the flagellated cells leave the wall and
sink into the central cavity, and undergoing certain changes establish
an inner mass of future dermal cells. By subsequent metamorphosis
the remaining flagellated cells become internal, not this time by
invagination, but by the included dermal cells breaking through the
wall of the larva, and forming themselves into a layer at the outside.

Fig. 112.—Median longitudinal section of parenchymula larva of Clathrina blanca.

p.g.c, Posterior granular cells—archaeocyte mother-cells. (After Minchin.)

In the larva of C. blanca, after a period of free-swimming existence,

the same three elements are thus recognisable as in that of Sycon at
the time of hatching; in the newly hatched larva of C. blanca,
however, one set of elements, the dermal cells, are not
distinguishable. The difference, then, between the two newly
hatched larvae is due to the earlier cell differentiation of the Sycon
larva.[254]

Now consider the larva of Leucosolenia. It is hatched as a

completely flagellated larva; its archaeocytes are internal (as in
Sycon); future dermal cells, recognisable as such, are absent. They
arise, as in C. blanca, by transformation of flagellated cells; but (1)
this process is confined to the posterior pole, and (2) the internal
cavity is small and filled up with archaeocytes. Consequently the
cells which have lost their flagella and become converted into dermal
cells cannot sink in as in C. blanca: they accumulate at the hinder
pole, and thus arises a larva half flagellated, half not; in fact, an
amphiblastula. Or, briefly, in Leucosolenia the larva at hatching is a
parenchymula, and when ready to fix is an amphiblastula; and,
again, the difference between the newly hatched larva and that of
Sycon is due to the earlier occurrence of cell differentiation in the
latter. What completer transitional series could be desired?

Turning to the Micromastictora, the developmental history already

sketched is fairly typical (p. 172). The differences between Mega-
and Micro-mastictoran larvae are referable mainly to the fact that the
dermal cells in the latter become at once differentiated among
themselves to form the main types of dermal cell of the adult.[255]
The metamorphosis is comparable to that of C. blanca. Among
Tetractinellida and Hexactinellida sexually produced larvae have not
been certainly identified.

Asexual reproduction takes place according to one of three types,

which may be alluded to as (1) "budding," (2) "gemmulation," (3)
formation of "asexual larvae."

By budding (Fig. 113) is meant the formation of reproductive bodies,

each of which contains differentiated elements of the various classes
found in the parent. A simple example of this is described by
Miklucho Maclay in Ascons, where the bud is merely the end of one
of the Ascon tubes which becomes pinched off and so set free.

In Leucosolenia botryoides[256] Vasseur describes a similar process;

in this, however, a strikingly distinctive feature is present (Fig. 114),
namely, the buds have an inverse orientation with respect to that of
the parent, so that the budding sponge presents a contrast to a
sponge in which multiplication of oscula has occurred. In fact, the
free distal end of the bud becomes the base of the young sponge,
and the osculum is formed at the opposite extremity, where the bud
is constricted from the parent.
 Fig. 113.—Lophocalyx philippensis. The specimen bears several buds attached
to it by long tufts of spicules. (After F. E. Schulze.)

Fig. 114.—Leucosolenia botryoides. A, a piece of the Sponge laden with buds, a-

f; i, the spicules of the buds directed away from their free ends; k, the
spicules of the parent directed towards the osculum, j. B, a bud which has
been set free and has become fixed by the extremity which was free or
distal in A. (After Vasseur.)

Such a reversal of the position of the bud is noteworthy in view of its

rarity, and the case is worth reinvestigating, for in other animal
groups a bud or a regenerated part retains so constantly the same
orientation as the parent that Loeb,[257] after experimenting on the
regeneration of Coelenterata and other forms, concluded that a kind
of "polarity" existed in the tissues of certain animals.

In Oscarella lobularis[258] the buds are transparent floating bladders,

derived from little prominences on the surface of the sponge.
Scattered in the walls of the bladders are flagellated chambers,
which open into the central cavity. The vesicular nature of the buds is
doubtless an adaptation, lessening their specific gravity and so
enabling them to float to a distance from the parent.

Gemmulation.—Spongilla has already afforded us a typical example

of this process. Gemmules very similar to those of Spongilla are
known in a few marine sponges, especially in Suberites and in
Ficulina. They form a layer attached to the surface of support of the
sponge—a layer which may be single or double, or even three or
four tiers deep. A micropyle is sometimes present in the spongin
coat, sometimes absent; possibly its absence may be correlated with
the piling of one layer of gemmules on another, as this, by covering
up the micropyle, would of course render it useless. Presumably
when a micropyle is present the living contents escape through it
and leave the sponge by way of the canal system (Fig. 115).

Fig. 115.—Gemmules of Ficulina. A, vertical section of gemmules in situ; B,

vertical section of upper portion of one gemmule. m, Micropyle.

The only case besides Spongilla in which the details of development

from gemmules have been traced is that of Tethya.[259] Mere
microscopic examination of a Tethya in active reproduction would
suggest that the process was simple budding, but Maas has shown
that the offspring arise from groups of archaeocytes in the cortex,
that is to say, they are typical gemmules. As they develop they
migrate outwards along the radial spicule-bundles and are finally
freed, like the buds of the Hexactinellid Lophocalyx (Fig. 113).

The comparison of the process of development on the one hand by

gemmules, and on the other by larval development, is of some
interest.[260] In both cases two cell layers—a dermal and a gastral—
are established before the young sponge has reached a functional
state. Differences of detail in the formation of the chambers occur in
the gemmule; these find parallels in the differences in the same
process exhibited by the larvae of various groups of sponges. On the
other hand, the order of tissue differentiation is not the same in the
gemmule as in the larva.

Fig. 116.—Development of the triradiate and quadriradiate spicules of Clathrina.

(1) Three scleroblasts; (2) each has divided: the spicule is seen in their
midst; (3) addition of the fourth ray by a porocyte. p, Dermal aperture of
pore; r, fourth ray. (After Minchin.)

Of the reproduction of Tetractinellida extremely little is known.

Spermatozoa occur in the tissues in profusion and are doubtless
functional, but larvae have been seldom observed.

Fig. 117.—Three stages in the development of the triradiate spicules of Sycon

setosum. × 1200. (After Maas.)

In Hexactinellida the place of sexually produced larvae is taken by

bodies of similar origin to gemmules but with the appearance of
parenchymulae. Ijima has indeed seen a few egg-cells in
Hexactinellids.[261] He finds, however, that archaeocyte congeries
occur in abundance, and there is good reason to believe with him
that these are responsible for the numerous parenchymula-like
asexually produced larvae he has observed. The discovery of
"asexual larvae" was first made by Wilson in the Monaxonid
Esperella; in this case the asexual larva is, as far as can be
detected, identical with that developed from the fertilised egg. A
similar phenomenon, the production of apparently identical larvae by
both sexual and asexual methods, has been observed in the
Coelenterate Gonionema murbachii.[262]

Artificially, sponges may be reproduced with great advantage to

commerce by means of cuttings. Cuttings of the bath sponge are fit
to gather after a seven years' growth.

The development of the various forms of spicules is a subject about

which little is yet known. Most spicules of which the development has
been traced originate in a single dermal cell. The triradiate and
quadriradiate spicules of Homocoela (Clathrinidae), as Minchin[263]
has most beautifully shown, form an exception. Three cells co-
operate to form the triradiate; these three divide to give six before
the growth of the spicule is complete. A quadriradiate is formed from
a triradiate spicule by addition of the fourth ray, which, again, has a
separate origin in an independent cell, in fact a porocyte. The
triradiate spicules of the Sycettidae, on the other hand, originate in a
single cell,[264] but the quadriradiate spicules are formed from these
by the addition of a fourth ray in a manner similar to that which has
just been described for Clathrinidae.
 Fig. 118.—Development of monaxon spicules. A, from Spongilla lacustris,
showing the single scleroblast. (After Evans.) B, a very large monaxon,
from Leucosolenia, on which many scleroblasts are at work. (After Maas.)

Monaxon spicules if not of large size undergo their entire

development within a single scleroblast (Fig. 118, A). In some cases
if their dimensions exceed certain limits, several cells take part in
their completion; some of these are derived from the division of the
original scleroblast, others are drawn from the surrounding tissue. In
Tethya, for example, and in Leucosolenia[265] the scleroblasts round
the large monaxon spicules are so numerous as to have an almost
epithelioid arrangement.

The large oxeas of Tetilla, Stelletta, and Geodia, however, are

formed each within a single scleroblast.[266]

Fig. 119.—Development of spheraster. A, of Tethya, from union of two

quadriradiate spicules. (After Maas.) B (a-e), of Chondrilla, from a spherical
globule. (After Keller.)
Triaenes have been shown[267] to originate as monaxons with one
swollen termination, from which later the cladi grow out. Information
as to the scleroblasts in this case is needed.

The value of a knowledge of the ontogeny of microscleres might be

great. Maas believes that he has shown that the spherasters of
Tethya are formed by the union of minute tetractine calthrops (Fig.
119, A). If this view should be confirmed, it would afford a very strong
argument for the Tetractinellid affinities of Tethya.

Fig. 120.—Stages in the development of the microscleres of Placospongia. (After

Keller.)

Keller,[268] on the other hand, finds that the spherasters of the

Tetractinellid Chondrilla originate as spheres (Fig. 119, B); and
spheres have been observed in the gemmule of a Tethya; no
spherasters were as yet present in the gemmule, and spheres were
absent in the adult.[269]

In the genus Placospongia certain spicules are present which

outwardly closely resemble the sterrasters so characteristic of
certain Tetractinellidae. Their development, however, as will be seen
from Fig. 120, shows that they are not polyaxon but spiny monaxon
spicules. Placospongia is consequently transferred to the
Monaxonida Spintharaphora.

Sterrasters originate within an oval cell as a number of hairlike

fibres[270] (trichites), which are united at their inner ends. The outer
ends become thickened and further modified. The position occupied
by the nucleus of the scleroblast is marked in the adult spicule by a
hilum.
 Fig. 121.—Three stages in the development of an anisochela. al, Ala; al', lower
ala; f, falx; f', lower falx; r, rostrum; r', lower rostrum. (After Vosmaer and
Pekelharing.)

The anisochela has been shown repeatedly to originate from a C-

shaped spicule.[271]

What little is known of the development of Hexactinellid spicules we

owe to Ijima.[272] Numerous cells are concerned in certain later
developmental stages of the hexaster; a hexaster passes through a
hexactin stage, and—a fact "possibly of importance for the
phylogeny of spicules in Hexactinellida"—in two species the first
formed spicules are a kind of hexactin, known as a "stauractin," and
possessing only four rays all in one plane (cf. Protospongia, p. 207).

Physiology
Production of the Current.—It is not at first sight obvious that the
lashing of flagella in chambers arranged as above described,
between an inhalant and an exhalant system of canals, will
necessarily produce a current passing inwards at the ostia and
outwards at the osculum. And the difficulty seems to be increased
when it is found[273] that the flagella in any one chamber do not
vibrate in concert, but that each keeps its own time. This, however, is
of less consequence than might seem to be the case. Two conditions
are essential to produce the observed results: (1) in order that the
water should escape at the mouth of the chamber there must be a
pressure within the chamber higher than that in the exhalant
passages; (2) in order that water may enter the chamber there must
be within it a pressure less than that in the inhalant passages. But
the pressure in the inhalant and exhalant passages is presumably
the same, at any rate before the current is started, therefore there
must be a difference of pressure within the chamber itself, and the
less pressure must be round the periphery. Such a distribution of
pressures would be set up if each flagellum caused a flow of water
directed away from its own cell and towards the centre of the
chamber; and this would be true whether the flagellum beats
synchronously with its fellows or not.

The comparative study of the canal systems of sponges[274]

acquires a greater interest in proportion as the hope of correlating
modifications with increase of efficiency seems to be realised. In a
few main issues this hope may be said to have been realised. The
points, so to speak, of a good canal system are (1) high oscular
velocity, which ensures rapid removal of waste products to a
wholesome distance; (2) a slow current without eddies in the
flagellated chambers, to allow of the choanocytes picking up food
particles (see below), and moreover to prevent injury to the delicate
collars of those cells; (3) a small area of choanocytes, and
consequent small expenditure of energy in current production.

It is then at once clear at what a disadvantage the Ascons are placed

as compared with other sponges having canal systems of the
second or third types. Their chamber and oscular currents can differ
but slightly, the difference being obtained merely by narrowing the
lumen of the distal extremity of the body to form the oscular rim.
Further, the choanocytes are acting on a volume of water which they
can only imperfectly control, and it is no doubt due to the necessity
of limiting the volume of water which the choanocytes have to set in
motion that the members of the Ascon family are so restricted in
size. The oscular rim is only a special case of a device adopted by
sponges at the very outset of their career, and retained and
perfected when they have reached their greatest heights; the volume
of water passing per second over every cross-section of the path of
the current is of course the same, therefore by narrowing the cross-
sectional area of the path at any point, the velocity of the current is
proportionally increased at that point. The lining of the oscular rim is
of pinacocytes; they determine a smooth surface, offering little
frictional resistance to the current, while choanocytes in the same
position would have been a hindrance, not only by setting up friction,
but by causing irregularities in the motion.

Canal systems of the second type show a double advance upon that
of the Ascons, namely, subdivision of the gastral cavity and much
greater length of the smooth walled exhalant passage. The
choanocytes have now a task more equal to their strength, and,
further, there is now a very great inequality between the total
sectional areas of the flagellated chambers and that of the oscular
tube.

Canal systems of the third type with tubular chambers are an

improvement on those of the second, in that the area of choanocytes
is increased by the pouching of the chamber-layer without
corresponding increase in the size of the sponge. However, the area
of choanocytes represents expenditure of energy, and the next
problem to be solved is how to retain the improved current and at the
same time to cut down expense. The first step is to change the form
of the chamber from tubular to spherical. Now the energy of all the
choanocytes is concentrated on the same small volume of water.
The area of choanocytes is less, but the end result is as good as
before. At the wide mouth of the spherical chamber there is
nevertheless still a cause of loss of energy in the form of eddies, and
it is as an obviation of these that one must regard the aphodi and
prosodi with which higher members of the Demospongiae are
provided. The correctness of this view receives support, apart from
mechanical principles, from the fact that the mass of the body of any
one of these sponges is greater relatively to the total flagellated area
than in those sponges with eurypylous chambers; that is to say, a
few aphodal and diplodal chambers are as efficient as many of the
eurypylous type.

It is manifest that the current is the bearer of the supply of food; but
it requires more care to discover (1) what is the nature of the food;
(2) by which of the cells bathed by the current the food is captured
and by which digested. The answer to the latter question has long
been sought by experimenters,[275] who supplied the living sponge
with finely powdered coloured matters, such as carmine, indigo,
charcoal, suspended in water. The results received conflicting
interpretations until it became recognised that it was essential to take
into account the length of time during which the sponge had been
fed before its tissues were subjected to microscopic examination.
Vosmaer and Pekelharing obtained the following facts: Spongilla
lacustris and Sycon ciliatum, when killed after feeding for from half
an hour to two hours with milk or carmine, contain these substances
in abundance in the bodies of the choanocytes and to a slight degree
in the deeper cells of the dermal tissue; after feeding for twenty-four
hours the proportions are reversed, and if a period of existence in
water uncharged with carmine intervenes between the long feed and
death then the chambers are completely free from carmine. These
are perhaps the most conclusive experiments yet described, and
they show that the choanocytes ingest solid particles and that the
amoeboid cells of the dermal layer receive the ingested matter from
them. In all probability it is fair to argue from these facts that solid
particles of matter suitable to form food for the sponge are similarly
dealt with by it and undergo digestion in the dermal cells.

Choanocytes are the feeding organs par excellence; but the

pinacocytes perform a small share of the function of ingestion, and in
the higher sponges where the dermal tissue has acquired a great
bulk the share is perhaps increased.

In the above experiments is implied the tacit assumption that

sponges take their food in the form of finely divided solids.
Haeckel[276] states his opinion that they feed on solid particles
derived from decaying organisms, but that possibly decaying
substances in solution may eke out their diet. Loisel, in 1898,[277]
made a new departure in the field of experiment by feeding sponges
with coloured solutions, and obtained valuable results. Thus
solutions, if presented to the sponge in a state of extreme dilution,
are subjected to choice, some being absorbed, some rejected. When
absorbed they are accumulated in vacuoles within both dermal and
gastral cells, mixed solutions are separated into their constituents
and collected into separate vacuoles. In the vacuoles the solutions
may undergo change; Congo red becomes violet, the colour which it
assumes when treated with acid, and similarly blue litmus turns red.
The contents of the vacuoles, sometimes modified, sometimes not,
are poured out into the intercellular gelatinous matrix of the dermal
layer, whence they are removed partly by amoeboid cells, partly, so
Loisel thinks, by the action of the matrix itself. It adds to the value of
these observations to learn that Loisel kept a Spongilla supplied with
filtered spring-water, to which was added the filtered juice obtained
from another crushed sponge. This Spongilla lived and budded, and
was in good health at the end of ten days.

Movement.—Sponges are capable of locomotion only in the young

stage; in the adult the only signs of movement are the exhalant
current, and in some cases movements of contraction sufficiently
marked to be visible to the naked eye. Meresjkowsky was one of the
early observers of these movements. He mentions that he stimulated
a certain corticate Monaxonid sponge by means of a needle point: a
definite response to each prick inside the oscular rim was given by
the speedy contraction of the osculum.[278]

Pigments and Spicules.—Various reasons lead one to conclude

that the spicules have some function other than that of support and
defence, probably connected with metabolism. For the spicules are
cast off, sometimes in large numbers, to be replaced rapidly by new
ones, a process for which it is difficult to find an adequate
explanation if the spicules are regarded as merely skeletal and
defensive.[279] Potts remarks upon the striking profusion with which
spicules are secreted by developing Spongillids from water in which
the percentage of silica present must have been exceedingly small.
The young sponges climbed up the strands of spicules as they
formed them, leaving the lower parts behind and adding to the upper
ends.
Of the physiology of the pigments of sponges not much is yet known:
a useful summary of facts will be found in Von Fürth's text-book.[280]

Spongin.—Von Fürth[280] points out that this term is really a

collective one, seeing that the identity of the organic skeletal
substance of all sponge species is hardly to be assumed. Spongin is
remarkable for containing iodine. The amount of iodine present in
different sponges varies widely, reaching in certain tropical species
of the Aplysinidae and Spongidae the high figure 8 to 14 per cent.
Seaweeds which are specially rich in iodine contain only 1.5 to 1.6
per cent.

In view of the fact that iodine is a specific for croup, it is of interest to

observe that the old herb doctors for many centuries recognised the
bath sponge as a cure for that disease.
 Fig. 122.—The ordinals measure (i.) the number of species, a-f, and (ii.) the
number of stations, a'-f', at which successful hauls were made. The
abscissae measure the depth: thus at I. the depth is from 0 to 50 fathoms;
at II. from 51 to 200; at III. from 201 to 1000; at IV. from 1001 upwards. a, a',
are the curves for Sponges generally; b, b', for Monaxonida; c, c', for
Hexactinellida; d, d', for Tetractinellida; e, e', for Calcarea; f, f', for Ceratosa.

Distribution in Space.—All the larger groups of Sponges are

cosmopolitan. Each group has, however, its characteristic
bathymetrical range: the facts are best displayed by means of
curves, as in Fig. 122, which is based wholly on the results obtained
by the "Challenger" Expedition. The information as to littoral species
is consequently inadequate, and we have not the data requisite for
their discussion.

Sponges generally (a) and Monaxonida in particular (b) are more

generally distributed in water of depths of 51 to 200 fathoms than in
depths of less than 50 fathoms; but localities in shallow water are
richer, for the station curve (a') rises abruptly from I. to II., while the
species curve (a) in the same region is almost horizontal.

The Hexactinellid curve (c) culminates on III., showing that the group
is characteristically deep water. That for Tetractinellida (d) reaches
its greatest height on II., i.e. between 51 and 200 fathoms. Even