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Modern Soil Microbiology
Third Edition
Modern Soil Microbiology
Third Edition

Edited by
Jan Dirk van Elsas, Jack T. Trevors,
Alexandre Soares ­Rosado, and Paolo Nannipieri
Cover images courtesy of:
Professor Emeritus Jan Dirk van Elsas, University of Groningen, Netherlands (upper picture); Professor Emeritus
Dr. George Barron, University of Guelph, Canada (left and right lower picture inserts); and Professor Raquel Peixoto,
Federal University of Rio de Janeiro, Brazil (middle lower picture insert).

CRC Press
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Library of Congress Cataloging-in-Publication Data

Names: Elsas, J. D. van (Jan D.), 1951- editor.

Title: Modern soil microbiology / editors: Jan Dirk van Elsas, Jack T.
Trevors, Alexandre Soares Rosado, Paolo Nannipieri.
Description: Third edition. | Boca Raton : Taylor & Francis, 2019.
Identifiers: LCCN 2018050977 | ISBN 9781498763530 (hardback : alk. paper) |
ISBN 9780429607929 (pdf) | ISBN 9780429602405 (epub) | ISBN 9780429596889
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Subjects: LCSH: Soil microbiology. | Molecular microbiology.
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Contents
Preface...............................................................................................................................................ix
Editors................................................................................................................................................xi
Contributors.................................................................................................................................... xiii

Section I Fundamental Chapters

Chapter 1 The Soil Environment...................................................................................................3

Jan Dirk van Elsas

Chapter 2 The Seven Grand Questions on Soil Microbiology (Selman A. Waksman,

Reexamined by Arthur D. McLaren).......................................................................... 21
Jan Dirk van Elsas and Paolo Nannipieri

Chapter 3 The Soil Microbiome—An Overview......................................................................... 37

Francisco Dini-Andreote and Jan Dirk van Elsas

Chapter 4 The Bacteria and Archaea in Soil............................................................................... 49

Jan Dirk van Elsas, Anton Hartmann, Michael Schloter, Jack T. Trevors, and
Janet K. Jansson

Chapter 5 The Fungi in Soil......................................................................................................... 65

Roger D. Finlay and R. Greg Thorn

Chapter 6 The Viruses in Soil—Potential Roles, Activities, and Impacts.................................. 91

Akbar Adjie Pratama and Jan Dirk van Elsas

Chapter 7 Horizontal Gene Transfer and Microevolution in Soil.............................................. 105

Kaare Magne Nielsen and Jan Dirk van Elsas

Chapter 8 The Protists in Soil—A Token of Untold Eukaryotic Diversity............................... 125

Michael Bonkowski, Kenneth Dumack, and Anna Maria Fiore-Donno

Chapter 9 Microbial Interactions in Soil................................................................................... 141

Jan Dirk van Elsas, Akbar Adjie Pratama, Welington Luis de Araujo, and
Jack T. Trevors

v
vi Contents

Chapter 10 Plant-Associated Bacteria and the Rhizosphere....................................................... 163

Abdul Samad, Günter Brader, Nikolaus Pfaffenbichler, and Angela Sessitsch

Chapter 11 Microorganisms Cycling Soil Nutrients.................................................................... 179

Penny R. Hirsch

Section II Methods Chapters

Chapter 12 Methods to Determine Bacterial Abundance, Localization, and

General Metabolic Activity in Soil........................................................................... 195
Lise Bonnichsen, Nanna Bygvraa Svenningsen, Mette Haubjerg Nicolaisen,
and Ole Nybroe

Chapter 13 Soil Microbiome Data Analysis................................................................................ 215

Francisco Dini-Andreote

Chapter 14 Soil Metagenomics: Deciphering the Soil Microbial Gene Pool.............................. 227
Sara Sjöling, Jan Dirk van Elsas, Francisco Dini Andreote, and
Jorge L. Mazza Rodrigues

Chapter 15 Analysis of Transcriptomes to Assess Expression and Activity Patterns

of the Soil Microbiome.............................................................................................. 245
Stefanie Schulz, Anne Schöler, Michael Schloter, and Shilpi Sharma

Chapter 16 Metaproteomics of Soil Microbial Communities...................................................... 257

Paolo Nannipieri, L. Giagnoni, and G. Renella

Chapter 17 Stable Isotope Probing—Detection of Active Microbes in Soil............................... 269

Marie E. Kroeger and Klaus Nüsslein

Chapter 18 Isolation of Uncultured Bacteria............................................................................... 295

Ulisses Nunes da Rocha

Chapter 19 Statistical Analyses of Microbiological and Environmental Data............................307

Alexander V. Semenov

Section III Applied Chapters

Chapter 20 Soil Microbial Communities and Global Change..................................................... 331

Mark P. Waldrop and Courtney Creamer
Contents vii

Chapter 21 Soil Suppressiveness to Plant Diseases..................................................................... 343

Christian Steinberg, Véronique Edel-Hermann, Claude Alabouvette, and
Philippe Lemanceau

Chapter 22 Plant Growth-Promoting Bacteria in Agricultural and Stressed Soils..................... 361

Elisa Gamalero and Bernard R. Glick

Chapter 23 Biodegradation and Bioremediation of Organic Pollutants in Soil........................... 381

Kam Tin Leung, Zi-Hua Jiang, Nouf Almzene, Kanavillil Nandakumar,
Kurissery Sreekumari, and Jack T. Trevors

Chapter 24 The Impact of Metal Contamination on Soil Microbial Community Dynamics......403

David C. Gillan and Rob Van Houdt

Chapter 25 Management Strategies for Soil Used for Cultivation, Including Modulation of
the Soil Microbiome.................................................................................................. 421
Alexandre Soares Rosado, Paolo Nannipieri, and Jan Dirk van Elsas
Glossary......................................................................................................................................... 431
Index............................................................................................................................................... 459
Preface
“Essentially, all life depends upon the soil. There can be no life without soil and no soil without life;
they have evolved together.” (Charles E. Kellogg, 1938).

Living soils are indispensable for human persistence as they are the environments where key bio-
geochemical processes take place. Such life-support processes, for example, the cycling of carbon,
nitrogen, sulfur, and phosphorus, are primarily carried out by different members of the soil microbi-
omes. Moreover, soil microbiomes are useful given their potential capacities to produce compounds
that are important for biotechnology, industry and medicine.
In light of its relevance for the functioning of Earth’s ecosystem, soil microbiology has been
termed one of the last “frontiers” in science.
Over the last decade, the study of soil microbiology has undergone significant changes, with
respect to both the understanding of the diversity and functioning of soil microbial communities
and the methods that are being used to dissect soil microbiomes into their components. In particu-
lar, the rapid development and use of DNA- or RNA-based molecular methods, giving rise to soil
metagenomics and soil metatranscriptomics, respectively, have revolutionized our knowledge of
the living soils across the globe. Moreover, soil proteomics has also undergone significant break-
throughs. Finally, the current capacities to generate big “molecular” soil data and analyze them using
advanced algorithms and deep learning approaches have also enabled unprecedented advances in
soil microbiology. In 2012, Paul and Nannipieri suggested that these important advancements have
given rise to an emergent “second golden age of soil microbiology.” The previous “golden age”
of soil microbiology was named by Waksman in 1932 and reflected the important discoveries of
diverse soil microorganisms being involved in key steps of the soil decomposition processes and of
the nitrogen and sulfur cycles.
What are the current advances about? Several instances of outstanding breakthroughs come to
mind. We have an improved knowledge of the acidobacteria and their diversity as major constitu-
ents of many terrestrial habitats. In addition, the role of archaea, previously believed to encompass
mainly extremophiles, in soil systems, particularly in nitrogen transformations (ammonia oxida-
tion), is becoming increasingly better understood. We also have an increased understanding of the
significance and drivers of the astounding microbial diversity in soils and the relevance of this
diversity for the mechanism of suppressing plant disease. The wealth of diverse niches in soil has
given rise to a range of diversification processes, and we now appreciate the importance of the key
horizontal gene transfer (HGT) processes in soil bacterial genome adaptations.
This book, Modern Soil Microbiology, Third edition, constitutes a thorough update of the highly
successful second edition. It covers all relevant topics in soil microbiology, with the aim to provide
a broad understanding of this fascinating interdisciplinary area that encompasses aspects of soil sci-
ence, ecology, physiology, genetics, molecular biology, biotechnology, biochemistry, and biophysics.
The book is divided into three sections that go from the fundamental, methods, to the applied side.
After depicting the soil and examining the seven grand questions posed by Selman A. Waksman,
as reexamined by Arthur D. McLaren, Section I addresses the aspects of soil as the habitat (matrix)
for microorganisms, describing the different organismal groups in it, including the bacteria, archaea,
viruses, fungi, and protozoa. The section examines their diversities and adaptive responses, as well
as their functioning in interactive and functional terms.
Section II describes the state of the art methods currently used in soil microbiology, examining
the novel knowledge these have provided and will continue to do so. Thus, traditional soil micro-
biology methods, next to soil metagenomics, soil metatranscriptomics, and soil metaproteomics
methods; stable-isotope probing, and novel methods of culturing are examined. Moreover, the use of
appropriate statistical methods, next to machine learning approaches, in soil analyses is examined.

ix
x Preface

Section III focuses on a range of applied aspects of soil microbiology, including the effects of
global warming, the nature of disease-suppressive soils, the use of biological control, biopesticides,
bioremediation, and heavy metal stresses in soil. Finally, approaches to modulate soil microbiomes
in production systems are discussed.
Modern Soil Microbiology will serve as a basic book for use in courses that aim to capture the
current developments in this discipline. It will be useful as a basic work that provides research sci-
entists with a quick entry into the specific topics and the most relevant literature.
The editors sincerely thank all authors and the publisher for their excellent cooperation and
contributions to this text. Special thanks are due to Akbar Adjie Pratama for his contributions to
several illustrations. As with all scientific and scholarly pursuits, we have expanded our knowledge
based on the discoveries of past generations of dedicated scholars, such as Selman A. Waksman and
Arthur D. McLaren. To them and other scholars, all of humanity is indebted. Our wish is that pres-
ent and future generations will use the science knowledge in this text for the benefit of humanity.
We welcome any comments from all who use this text.

Jan Dirk van Elsas

Jack T. Trevors
Alexandre Soares Rosado
Paolo Nannipieri
Editors
Jan Dirk van Elsas is an emeritus professor of microbial ecology at the University of Groningen,
Groningen, the Netherlands. He obtained his MSc degree in chemical technology at the University
of Delft, Delft, the Netherlands, and then earned his PhD degree in microbiology from the Institute
of Microbiology Professor Paulo de Góes at the Federal University of Rio de Janeiro (IMPPG-
UFRJ), Rio de Janeiro, Brazil. From Rio de Janeiro, he moved to Wageningen University and
Research Centre, Wageningen, the Netherlands, where he occupied various positions, lastly as the
leader of the Microbial Buffering group at the Plant Research International Institute. In 2003, he
moved to the University of Groningen, Groningen, the Netherlands, where he was appointed to the
chair professorship of Microbial Ecology. He is a specialist on bacterial survival and evolution by
horizontal gene transfer in soil, the rhizosphere, and the mycosphere. He has published more than
300 peer-reviewed papers in this area and edited several books. He has been a leader in the explora-
tion of horizontal gene processes and agents in soil activity hot spots, including the rhizosphere and
mycosphere. He formally retired in 2017 and is currently involved in a range of science policy and
management activities.

Jack T. Trevors is an emeritus professor of microbiology at the University of Guelph, Guelph,
Ontario, Canada. He obtained his BSc (biology) and MSc (microbiology) degrees from Acadia
University, Wolfville, Nova Scotia, Canada, and his PhD degree (microbiology) from the University
of Waterloo, Waterloo, Ontario, Canada. His academic career was at the University of Guelph,
Guelph, Ontario, Canada, from 1982 to 2012. His areas of expertise are applied and environmental
microbiology, bioremediation, pathogens in the environment, gene transfer, bacterial ­survival and
activities in the environment, gene expression, microbiological methods, basic cell biology and the
origin and evolution of microorganisms. Professor Trevors has published over 450 publications,
­co-edited several books, has been a highly cited researcher, is a member of several science acad-
emies and is currently the editor-in-chief of the Journal of Microbiological Methods and Water, Air
and Soil Pollution. He is also an editor with the journal Antonie van Leeuwenhoek and the book
series editor for Environmental Pollution.

Alexandre Soares Rosado is currently a professor at the Federal University of Rio de Janeiro
(UFRJ), Rio de Janeiro, Brazil, and is also a visiting professor at the Department of Land, Air,
and Water Resources, University of California, Davis, California. He is the former director of the
Institute of Microbiology Professor Paulo de Góes at UFRJ (IMPPG-UFRJ) and vice president of
the Brazilian Society of Microbiology. He holds a BSc in biological sciences, an MSc in microbi-
ology, and a PhD in microbiology from UFRJ. This included a sandwich period at Wageningen
University and Research Centre, Wageningen, the Netherlands. Professor Rosado is an environ-
mental microbiologist specializing in the molecular ecology of soil, extreme environments, and
bioremediation.

Paolo Nannipieri is an emeritus professor at the University of Florence, Florence, Italy. He obtained
the degree in biological sciences in December 1969, was a researcher at the Institute for Soil
Chemistry, National Research Council, Pisa (1972–1986), and occupied the chair of Soil Chemistry
at the Faculty of Agriculture at the University of Tuscia, Viterbo, Italy (1986–1990) and the chair
of Agricultural Biochemistry at the Faculty of Agriculture at the University of Florence, Florence,
Italy. In the latter Institute, he was head of the Department of Agrifood and Environmental Sciences

xi
xii Editors

and the Department of Agrifood Production and Environmental Sciences. He is the author and
coauthor of about 250 publications (one in Nature), mostly in international scientific journals, and
the editor of several books. He is the editor-in-chief of Biology and Fertility of Soils. He received
the Lifetime Achievement Award “Terrestrial Enzymology” during the meeting “Enzymes in the
Environment—Ecology, Activity & Applications,” Bangor, Wales, July 26, 2016. He has been a
highly-cited researcher in both 2015 (among 44 Italian researchers) and 2016, a­ ccording to Thomson
Reuters. Professor Nannipieri is a specialist of Soil Biochemistry.
Contributors
Claude Alabouvette Kenneth Dumack
Agroécologie, AgroSup Dijon, CNRS, INRA Cluster of Excellence on Plant Sciences
University of Bourgogne Franche-Comté (CEPLAS)
Dijon, France Terrestrial Ecology Group, Institute of Zoology
University of Cologne
Nouf Almzene Köln, Germany
Department of Chemistry, Lakehead University
(Thunder Bay Campus), Véronique Edel-Hermann
Ontario, Canada Agroécologie, AgroSup Dijon, CNRS, INRA
University of Bourgogne Franche-Comté
Welington Luis de Araújo Dijon, France
Department of Microbiology
Institute of Biomedical Sciences Jan Dirk van Elsas
University of São Paulo Department of Microbial Ecology
São Paulo, Brazil GELIFES
University of Groningen
Michael Bonkowski The Netherlands
Cluster of Excellence on Plant Sciences
(CEPLAS) Roger D. Finlay
Terrestrial Ecology Group, Institute of Zoology Department of Forest Mycology & Pathology
University of Cologne Uppsala BioCenter, Swedish University of
Köln, Germany Agricultural Sciences
Uppsala, Sweden
Lise Bonnichsen
Department of Plant and Environmental Anna Maria Fiore-Donno
Sciences Cluster of Excellence on Plant Sciences
Faculty of Science (CEPLAS)
University of Copenhagen Terrestrial Ecology Group, Institute of
Frederiksberg, Denmark Zoology
University of Cologne
Günter Brader Köln, Germany
AIT Austrian Institute of Technology
Center for Health and Bioresources, Elisa Gamalero
Bioresources Unit Dipartimento di Scienze e Innovazione
Tulln, Austria Tecnologica
Università del Piemonte Orientale
Courtney Creamer Alessandria, Italy
U.S. Geological Survey
Menlo Park, California L. Giagnoni
Department of Agrifood Production and
Francisco Dini-Andreote Environmental Sciences
Department of Microbial Ecology University of Florence
Netherlands Institute of Ecology Florence, Italy
(NIOO-KNAW)
Wageningen, the Netherlands

xiii
xiv Contributors

David C. Gillan Paolo Nannipieri

Proteomics and Microbiology Lab, Bioscience Department of Agrifood Production and
Institute Environmental Sciences
Mons University University of Florence
Mons, Belgium Florence, Italy

Bernard R. Glick Mette Haubjerg Nicolaisen

Department of Biology Department of Plant and Environmental Sciences
University of Waterloo Faculty of Science
Waterloo, Ontario, Canada University of Copenhagen
Frederiksberg, Denmark
Anton Hartmann
Helmholtz Zentrum München Kaare Magnus Nielsen
Department of Environmental Sciences Department of Life Sciences and Health
Neuherberg/Munich, Germany Oslo Metropolitan University
Oslo, Norway
Penny R. Hirsch
Rothamsted Research Klaus Nüsslein
Harpenden, United Kingdom Department of Microbiology
University of Massachusetts Amherst
Rob Van Houdt Amherst, Massachusetts
Microbiology Unit, Interdisciplinary
Biosciences Ole Nybroe
Belgian Nuclear Research Centre (SCK•CEN) Department of Plant and Environmental Sciences
Mol, Belgium Faculty of Science
University of Copenhagen
Janet K. Jansson Frederiksberg, Denmark
Pacific Northwest National Laboratory
Washington Nikolaus Pfaffenbichler
AIT Austrian Institute of Technology
Zi-Hua Jiang Center for Health and Bioresources,
Lakehead University (Thunder Bay Campus) Bioresources Unit
Thunder Bay, Ontario, Canada Tulln, Austria

Marie E. Kroeger Akbar Adjie Pratama

Department of Microbiology Department of Microbial Ecology,
University of Massachusetts Amherst Microbial Ecology
Amherst, Massachusetts Groningen Institute for Evolutionary Life
Sciences, University of Groningen
Philippe Lemanceau Groningen, the Netherlands
Agroécologie, AgroSup Dijon, CNRS, INRA
University of Bourgogne Franche-Comté G. Renella
Dijon, France Department of Agrifood Production and
Environmental Sciences
Kam Tin Leung University of Florence
Lakehead University (Thunder Bay Campus) Florence, Italy
Thunder Bay, Ontario, Canada
Ulisses Nunes da Rocha
Kanavillil Nandakumar Helmholtz Centre for Environmental
Lakehead University (Orillia Campus) Research—UFZ
Orillia, Ontario, Canada Leipzig, Germany
Contributors xv

Jorge L. Mazza Rodrigues Shilpi Sharma

Department of Land, Air and Water Resources Indian Institute of Technology
University of California, Davis Department of Biochemical Engineering and
Davis, California Biotechnology
Delhi, New Delhi, India
Alexandre Soares Rosado
Federal University of Rio de Janeiro Sara Sjöling
Rio de Janeiro, Brazil Environmental Science, School of Natural
Sciences
Abdul Samad Technology and Environmental Studies
AIT Austrian Institute of Technology Södertörn University
Center for Health and Bioresources, Huddinge, Sweden
Bioresources Unit
Tulln, Austria Kurissery Sreekumari
Lakehead University (Orillia Campus)
Michael Schloter Orillia, Ontario, Canada
Helmholtz Zentrum München
Research Unit for Comparative Microbiome Christian Steinberg
Analysis Agroécologie, AgroSup Dijon, CNRS, INRA
Department of Environmental Sciences University of Bourgogne Franche-Comté
Neuherberg/Munich, Germany Dijon, France

Anne Schöler Nanna Bygvraa Svenningsen

Helmholtz Zentrum München Department of Plant and Environmental
Research Unit for Comparative Microbiome Sciences
Analysis Faculty of Science
Department of Environmental Sciences University of Copenhagen
Neuherberg, Germany Frederiksberg, Denmark

Stefanie Schulz R. Greg Thorn

Helmholtz Zentrum München Department of Biology
Research Unit for Comparative Microbiome University of Western Ontario
Analysis London, Ontario, Canada
Department of Environmental Sciences
Neuherberg, Germany Jack T. Trevors
School of Environmental Sciences
Alexander V. Semenov University of Guelph
Incotec Europe BV Guelph, Ontario, Canada
Enkhuizen, the Netherlands
Mark P. Waldrop
Angela Sessitsch U.S. Geological Survey
AIT Austrian Institute of Technology Menlo Park, California
Center for Health and Bioresources,
Bioresources Unit
Tulln, Austria
Section I
Fundamental Chapters
 1 The Soil Environment*
Jan Dirk van Elsas
University of Groningen

CONTENTS
1.1 Introduction...............................................................................................................................3
1.2 Scales and Gradients..................................................................................................................4
1.2.1 Introduction...................................................................................................................4
1.2.2 Spatial and Temporal Scales in Soil..............................................................................5
1.3 The Soil Physicochemical Environment....................................................................................6
1.3.1 Water—The Essential Factor for Soil Life....................................................................6
1.3.1.1 Definitions.......................................................................................................8
1.3.2 Soil as an Energy and Nutrient Source..........................................................................9
1.3.3 Soil Temperature.......................................................................................................... 11
1.3.4 Soil Light..................................................................................................................... 14
1.3.5 Soil Atmosphere and Redox Potential......................................................................... 14
1.3.6 Soil pH......................................................................................................................... 16
1.4 Concluding Remarks............................................................................................................... 18
References......................................................................................................................................... 18

1.1 INTRODUCTION
Soil is a structured environment that “holds” a wealth of organisms with diverse activities and func-
tions. Among these organisms, microorganisms have a central place, as they play major roles in key
soil processes. However, a key problem in any discussion about soil as a microbiological habitat is
our conceptual visualization of soil. The most commonly used unit of reference is 1 g. This unit has
been an optimum mass from which biodiversity indices indicative of habitat richness are gained.
One gram of soil consists of inorganic and organic fractions. The inorganic particles of soil are clas-
sified into three major groups according to their size: sand, silt, and clay. The proportions of these
in any soil determine the soil texture. The number of particles in 1 g of soil can range from 90 (pure
coarse sand) to 90 billion (pure clay), and the proportions of sand, silt, and clay in the soil sample
determine the soil textural class. Assuming spherical shapes, the surface area of soil particles can
range from 11 to 8 million cm2 g−1, illustrating the high degree of physical heterogeneity present in
any given soil sample. Although the concept of an “average” soil is not entirely meaningful, we will
consider a hypothetical soil aggregate (Figure 1.1) as the basic unit of the soil habitat. Many biogeo-
chemical processes occur at scales more or less relevant to this unit, including processes such as gas
diffusion and water movement, which create a mosaic of microsites and gradients (Paul and Clark
1996). Most, if not all, aggregates constitute potential habitats that allow the survival of one or more
species of soil microorganisms. In our “hypothetical” habitat, a gram of soil and the arrangement
of the soil particles (soil structure; see Table 1.1) are vital to the microbes present. About half the

* Modified from Standing and Killham (2007).

3
4 Modern Soil Microbiology

Zymogenous heterotrophs,
fast growing on organic
(obligate requirement for O2) substrate
Aerobic microorganisms
Facultative Autochthonous
(adaptation to heterotrophs, slow, steady
+/- oxygen growth on organic substrate
stress)
Chemoautotrophs, energy
Anaerobic microorganisms via oxidation of compounds
(obligate requirement for O2
absence)

2 mm
Oxygen

N2, NO2, CO2

pH

Soil water

Predation

FIGURE 1.1 Schematic of a section through a 2 mm-diameter soil aggregate microsite yielding habitats
for bacteria based on physiological requirements. The arrows show the main directions of diffusion for key
processes. The increasing darkness of the soil particles indicates the increasing number of sites available for
anaerobic bacterial function.

volume of an aggregate will be void spaces (pores) connected by tortuous pathways presenting a
range of pore neck sizes. It is the relationship between the interconnecting pathways, channels, and
pores in soil that provides the microhabitat space (niche, used here as the term to describe habit-
able space in soil) for the soil microbiota. The physical niches themselves will mainly be soil pore
walls, although water in channels may contain, and transport, significant numbers of freely motile
bacteria (Figure 1.2).
Soil water passes more rapidly through wider pores by means of gravity mass flow and diffusion
(see below for a separate consideration of these topics) than through narrow pores. The physical
niches in the wider pores are also mainly pore walls to which bacteria adhere.

1.2 SCALES AND GRADIENTS

1.2.1 Introduction
The microbial habitat in soil is, essentially, a porous medium that varies both spatially and tem-
porally. The structural form of this porous medium is the product of many different processes.
Biological, physicochemical, and mechanical processes act together to aggregate, compact, crack,
and fragment the soil, resulting in a soil structure consisting of solids and pores. The soil pores form
a tripartite group—transmission pores being the main conduits for water and nutrient flow, storage
pores that are empty under the influence of matric potential, and residual pores (with pore neck
diameters less than 0.3 μm) that remain water-filled. In most soils, the transmission pores form the
majority of pore space in the soil microbial habitat.
The Soil Environment 5

TABLE 1.1
Approximate Dimensions (μm) of Soil Particles and Biota and Comparison of
Water-Filled Pores and Water Films
Type Approximate Dimensions (μm)
Soil particles Stones >2,000
Coarse sand 2,000–200
Fine sand 200–50
Silt 50–2
Clay 2–0.2
Plant material Roots 1,000
Fine roots 1,000–50
Root hairs 15–7
Microbes Fungal hyphae 10–3
Actinomycetes 1.5–1.0
Bacteria 0.5–1.0
Viruses 0.05–0.2
Some soil animals Earthworms 5,000–2
Mites 2,000–500
Nematodes 2,000–500
Protozoa 80–10
Water-filled pores −10 kPa <30
−100 kPa <3
−1,000 kPa 0.3
Water films −100 kPa <0.0003
−1,000 kPa Few molecules thick

Bacteria are highlighted in bold for comparison with other components of the soil–microbe–plant system.

Historically, it has proven to be impossible to produce an overarching descriptor of soil habitats

that fits all soil types (not to be confused with soil classification). It would be useful to have such
a descriptor, as it may help us in understanding the observable conservation of microbial genes
responsible for the processes carried out in soils, such as denitrification and carbon, nitrogen, and
phosphorus mineralization.
Now entering a bit of theory: fractals (objects with fractional dimensions which possess self-
similarity, composed of several parts, each of which is a small-scale copy of the whole) have been
applied successfully to describe spatiotemporal, hierarchical, and complex systems. These fractal
systems can be generated using scaling laws and iterative algorithms. To be classified as a fractal
system, patterns must be self-similar over a range of scales (i.e., its properties are reproduced at
a number of different spatial and temporal scales). As a result, no matter how intricate a particu-
lar pattern might be, its statistical properties allow its description, independent of scale (Bird and
Perrier 2003, Perrier and Bird 2002). Within a given soil texture (except for very clayey soils), a
description of soil as a system of fractals appears to be robust. However, high clay content soils do
not display fractal particle size distributions (Millan et al. 2003).

1.2.2 Spatial and Temporal Scales in Soil

Spatial and temporal scales in soil can be best understood by way of an example. Here, we use a
root with its surrounding soil to illustrate some key spatiotemporal aspects (Figure 1.3). The soil
6 Modern Soil Microbiology

FIGURE 1.2 Possible niche space available to bacteria (shown in black, adhering to soil particle (gray hatch-
ing) surfaces showing the relationship between soil matric potential and pore size available.

habitat generally constitutes an aerobic, oligotrophic (nutrient-poor) environment, which is not con-
ducive to high population densities and activities of microorganisms. In contrast, the ­rhizosphere
(further discussed in Chapter 10) provides an environment in which elevated population sizes and
activities of fast-growing (formerly called zymogenous) microorganisms are supported by plant-
derived carbon substrates, in the form of exudates and cell lysates. This constitutes the soil–plant
interface, a habitat that incites a wide spectrum of beneficial as well as detrimental associations with
microorganisms.
Water movement and diffusion of molecules are key features of the soil and rhizosphere habitats
in which microbial populations transmit information from one to another, for example, via quorum
sensing (Whiteley et al. 2017). Bacterial interactions that are important in the rhizosphere (e.g.,
production of antibiotics and chitinases, biofilm formation, stationary phase, and motility) (Cha
et al. 1998, Elasri et al. 2001, Pierson et al. 1998) are often driven by quorum sensing. See Chapters
9 and 10 for more detailed information about these interactions. Key issues arise here, such as the
connectivity of the soil pores, which determines to a great extent the local concentrations of quorum
sensing molecules. These concentrations ultimately determine the outcome of the quorum sensing-
dependent processes.

1.3 THE SOIL PHYSICOCHEMICAL ENVIRONMENT

1.3.1 Water—The Essential Factor for Soil Life
“Understanding the movement of water in soil is understanding the most significant feature of
the soil as a habitat for microbial life”. Where water moves, so do ions and nutrients. Water car-
ries dissolved gases and heat, and also bacteria and their predators. It protects microhabitats from
The Soil Environment 7

Soil aggregates
ROOT
SOIL SOIL

Diffusion of water and

nutrients to and from root,
and between aggregates

Growth Root exudates: carbon

compounds including substrates
and signals
Rhizobacterial population
sizes, typically 109 at root-
soil interface, decreasing
Scale 1 mm

FIGURE 1.3 Schematic illustration of the soil environment around plant roots in terms of diffusion of mate-
rials (substrate carbon and nutrients) and information (signal molecules). Scale: 1 mm

desiccation and opens other potential habitats while closing others. The fundamental relationships
between physics and chemistry that modulate soil water and biological activity are presented in the
schematic picture in Figure 1.4. When considering any interaction of soil water and biological activ-
ity, it must be stressed that the four central boxes (representing physical and chemical laws) cannot
be discussed in isolation: they all contribute to the interaction.

SOIL WATER
PHYSICAL and

co-activities
CHEMICAL

Nutrient Temperature
diffusion and Mobility and pH and Eh
mass flow Aeration

SOIL BIOLOGICAL ACTIVITY

FIGURE 1.4 The range of water-influenced soil properties and processes which determine the microbial
activity. Eh: redox potential. See Section 1.3.5.
8 Modern Soil Microbiology

1.3.1.1 Definitions
Soil water potential is the sum of the matric, osmotic, and pressure potentials; it is the key measure
of the activity of water in the soil. These three component terms are briefly outlined in the follow-
ing text. For a fuller consideration of these topics, see Smith and Mullins (2000) and Marshall and
Holmes (1996).
Matric potential: Water molecules adsorb onto the surface of soil minerals through hydrogen
bonding, as well as bonding cohesively with other water molecules. These adhesive and cohesive
forces act together to hold soil water under tension against external forces such as gravity. Because
of this, the soil water always has less potential energy than free water (reference water: at the same
temperature, pressure, and location as the soil water) and can never carry a positive sign. Thus,
matric potential is always negative or zero.
Osmotic potential: Soil water is not pure water but a solution containing varying amounts of
osmotically active organic molecules and inorganic salts, which decrease the potential energy of
soil water relative to a pure water reference. Thus, like matric potential, osmotic potential is always
negative or zero.
Pressure potential: The pressure potential component comes from external forces (including
gravity) exerted on soil water. In a flooded soil with a layer of standing water, the atmosphere as
well as the surface water exerts a positive pressure on the soil water. In the absence of flooding, it is
simply the pressure of the atmosphere alone, and this is the reference state. The additional pressure
of ponded water creates a positive pressure.
Water is held dynamically in the soil by forces that act to reduce the potential energy relative to
that of free water (at the same temperature, pressure, and location). The relationship of the water
potential to microbial habitat can be visualized in a moisture release curve when drying soil is con-
sidered (Figure 1.5). Water will be drained gravitationally out of large pores first, followed by drain-
age from successively smaller pores. It should be emphasized here that it is the pore neck diameter
that determines the rate of water movement, that is, a large water-filled pore with a small pore neck
diameter will empty slower than a small pore with a large pore neck diameter. As soils rewet, it is
not a simple reverse of drainage. Paradoxically, at a given suction force, a drying soil has higher
water content than a wetting soil. This phenomenon is termed hysteresis. It is a function of the pore
diameter generally being larger than the corresponding pore neck diameters; a full explanation is
available in the work of Marshall and Holmes (1996).
Increases in soil moisture content can greatly affect bacterial population dynamics by providing
connections between separate aggregates. Vargas and Hattori (1990), for example, showed that the
level of bacterial predation was sensitive to aggregate connectivity. In wet soils (>60% of water-
holding capacity), up to 90% of the bacteria present in the soil organic matter were consumed,
whereas this percentage was lower in drier soils. As already mentioned, the soil pore structure and
pore neck diameter allow the movement of water and, with the water, microorganisms through
soils. Without water movement, most microorganisms actually move poorly through soils (Yang
and van Elsas 2018). With respect to the water movement, it is the pore neck diameter that deter-
mines whether a pore is filled or unfilled at a particular matric potential (the largest water-filled pore
neck diameter estimated from soil water release curves as exemplified in Figure 1.5). The largest
water-filled pore neck diameters, at a particular matric potential, can be calculated from the simple
equation:

ψ = 300 d

where
ψ is the matric potential (−kPa)
d is the neck diameter of the largest water-filled pore (μm)
The Soil Environment 9

FIGURE 1.5 A hypothetical moisture release characteristic, demonstrating the relationship between water con-
tent, water potential, and neck diameter of the largest water-filled pore (Adapted from Gammack et al. 1991).

Thus, by applying a given matric potential to the soil, it is possible (with a finely nebulized inoculum
in a precise volume with reference to the moisture release curve of the soil) to preferentially fill pore
size classes (White et al. 1994). This elegant technique can be used to experimentally place bacteria
in particular pore size classes of choice or to explore such topics as the consequences of grazing
limitation (undergrazing of excluded pores and overgrazing of included pores) on community struc-
ture and function.

1.3.2 Soil as an Energy and Nutrient Source

Figure 1.6 shows how soil microorganisms are classified according to how they use their sources of
energy and carbon. Light is obviously only present at the surface of the soil. The chemical energy
for chemoautotrophs is supplied in the form of reduced inorganic chemicals, such as Fe2+, and
reduced sulfur or nitrogen compounds (e.g., SO32−, S2−, NH4+, and NO2−).
Organic matter provides the energy source for the heterotrophic microbial community of the
soil. This organic matter ranges from the readily decomposable and more resistant (“recalcitrant”)
fractions of plant litter to the fractions of microbially processed organic matter that are either
­physically or chemically protected to some degree. Most importantly, the microbial biomass itself
represents the “eye of the needle” through which all these fractions eventually pass (Figure 1.7). In
the figure, degradation-resistant plant material is typified by lignin (a complex polymer of aromatic,
10 Modern Soil Microbiology

SOIL MICROORGANISMS

Energy source
Light Chemical

Phototrophy Chemotrophy

CO2 Organic C C-source CO2 Organic C

Photolithotrophs Photoorganotrophs Chemolithotrophs Chemoorganotrophs

or or or or

Photoautotrophs Photoheterotrophs Chemoautootrophs Chemoheterotrophs

FIGURE 1.6 Classification of soil microorganisms in terms of use of carbon/energy sources.

i.e., cellulose i.e., lignin

DPM- (readily)
DPM RPM
decomposable plant
material
k = 4 y-1 k = 0.3 y-1

RPM- resistant plant

material
CO MICROBIAL
2 BIOMASS k = 0.4 y-1 POM- physically
protected organic matter

COM- Chemically
k = 0.01 y-1 k = 0.0003 y-1 protected organic matter

POM COM k- first-order decay

constants

FIGURE 1.7 Main redox couples (terminal electron acceptor and redox product) and associated microbial
processes operating in the soil system in relation to the redox potential at neutral pH.
The Soil Environment 11

phenyl propane, repeating units), whereas readily decomposable plant material is typified by cellulose
(carbohydrate polymer with repeating glucose units). In addition to soil organic matter, the organic
carbon that is directly released from plant roots represents a key energy source. This rhizosphere
carbon flow, which comprises C in a variety of forms, can account for a considerable part (5%–25%)
of the plant photosynthates (Lambers 1987); it is the main energy source for the heterotrophic
microbes, particularly bacteria, that colonize the rhizosphere.
Not only does soil organic matter represent an energy source which is vital to the heterotrophic
soil microorganisms, but it is also the main supplier of nutrients, as most of the N, P, and S reserves
of the soil are tied up in organically bound forms. Some of the microbial transformations (and the
associated enzymes) involved in the release of these bound nutrients are shown in the following text.
These all produce small and simple compounds such as ammonia, phosphate, and sulfate, which
can be directly used as nutrients by plants or microorganisms.
Deamination of amino acids (release of organically bound N)

COOH COOH
deaminase
— —

— —
H2N—C—H C—
— O + NH3

CH CH
—

CH —CH
—

—
COOH COOH

ketoglutamic acid
glutamic acid
+ammonia

Urease activity (release of organically bound N from urea)

CO ( NH 2 )2 + H 2 O → 2NH 3 + CO 2

Phosphatase activity (release of organically bound P from phosphate monoesters)

O O
—
— —

—
—

ROPOH + H20 ROH + HOPOH

—

OH OH

Sulfatase activity (release of organically bound S from sulfate monoesters)

ROSO3− + H 2 O → ROH + H + + SO4 2−

1.3.3 Soil Temperature
Temperature in soil is a key determinant of both the distribution and the activity of soil microorgan-
isms. In terms of activity, temperature directly affects microbial physiology, and it indirectly exerts
its effects through changes in factors such as nutrient and substrate diffusion and water activity.
Soil temperature is the product of incident solar energy, modified by a series of factors. The first
is reflectance (about one-third of the incident solar energy is reflected back by the soil–plant system,
leaving two-thirds of the “net irradiation”), which is determined by soil color and vegetation type.
The next factor is soil moisture. About 80% of the net radiation is used to evaporate water from the
soil compared to 5% which is used for photosynthesis. So, only about 15% of the net radiation tends
12 Modern Soil Microbiology

to warm the soil. The extent to which a unit of soil is warmed by a unit of net radiation is determined
by the specific heat capacity of the soil and the soil moisture (the latter is important because of the
specific heat capacity of water and the energy needed for the vaporization of water). In addition to
these fundamental principles, soil temperature regime is affected by seasonal factors, as well as
“edaphic” factors such as soil type/depth and the nature of the vegetation present.
Temperature is a strong selective force and is a key driver of physical, chemical, and physi-
ological reactions. As such, it is of significant importance in soil, and soil microbial communities
can be defined on the basis of temperature values alone (Figure 1.8). Within “average” mesophilic
microbial communities, there is an approximate doubling of the rate of biochemical activity with
every 10°C rise between 0°C and 30°C/35°C. This is referred to as the Q10 relationship. At the
soil surface, the temperature will be primarily affected by the incident short-wavelength radiation
(<2 μm), and it will fluctuate both diurnally and annually. These fluctuations have progressively less
amplitude from the surface to deeper layers in the soil (Figure 1.9).
From the Q10 relationship, we can deduce that soil temperature is a critical regulator of microbial
metabolism. Figure 1.9 illustrates how soil temperature can fluctuate with time and depth. How soil
microorganisms respond to changes in temperature is not, of course, independent of the effects of
temperature on the plants (and other organisms) with which they interact. For example, the quantity

Temperature (ºC)
-20 -10 0 10 20 30 40 50 60 80 90

Psychrophiles

Mesophiles
Thermophiles
Hyperthermophiles*

FIGURE 1.8 Schematic showing temperature preferences for soil bacteria.

* The majority of this group are archaea which are generally found in aqueous environments rather than soils.

Approximate upper temperature limit to microbial survival

Soil temperature, departure from mean

+12 40

+8 30
Soil temperature (ºC)

+4 20

0 10

-4 0

-8 -10

-12 -20
Approximate lower temperature limit to microbial survival

24 48 72 96
Time (hours)

FIGURE 1.9 Trend of diurnal temperature fluctuation in soil as related to depth. The periodicity of fluctua-
tion can be considered diurnally or seasonally.
The Soil Environment 13

and quality of rhizosphere C flow is critical to the diversity and activity of the rhizosphere microbial
community and is strongly dependent on temperature (Meharg and Killham 1989), as is root growth
and turnover.
Some soil microbial processes are particularly temperature sensitive. For instance, the accumu-
lation of ammonium in temperate–climate soils in autumn and spring is due to the marked differ-
ence in low-temperature sensitivity of the processes of ammonification and nitrification, as outlined
in the following scheme:

Ammonification (carried out Nitrification (carried out by

by a very diverse group of a very narrow range of
soil microorganisms) soil microorganisms)
Organic matter → NH +4 → NO3−
Selective low-temperature
block

Table 1.2 shows the classification of soil microorganisms on the basis of temperature preference.
Soil microorganisms are truly peculiar: some can live at temperatures that could not be imagined
by higher life forms (see Box 1.1) (Killham 1994).

TABLE 1.2
Classification of Soil Microorganisms According to Temperature Preference
Environmental Class Temperature Range (°C) Optimum Growth (°C)
Psychrophile −5 ~ 20 15
Mesophile 15 ~ 45 37
Thermophile Moderate 40 ~ 70 60
Extreme: (hyperthermophile) 65 ~ 95 85

BOX 1.1 EXTREME SOIL ENVIRONMENTS

That no region on Earth, with the possible exception of active volcanoes, is devoid of soil
microbial life, is truly amazing in terms of the temperature and pH tolerances and adapta-
tion of the microorganisms present. First, the thermophilic S-oxidizing bacteria and ther-
mophilic algae that inhabit hot springs (often close to the boiling point of water) on the one
hand, and the psychrophilic snow moulds that can decompose leaf litter below the snow
cover at virtually zero degrees centigrade on the other hand, give us an indication of life
across the spectrum of temperatures in our environments. This connects to pH tolerance
as well. An extreme example of physiological pH preference occurs in some of the S- and
Fe-oxidizing chemoautotrophic bacteria. Thiobacillus ferrooxidans and T. thiooxidans,
for example, grow optimally at pH 2-3, while the soil bacterium T. acidophilus can be
termed a true acidophile, being routinely cultured at pH 1.4. This is amazing, bearing in
mind the logarithmic relationship between pH and hydrogen ion concetration. It means
that T. acidophilus is growing at nearly a million times the acidity of many near-neutral
agricultural soils!
14 Modern Soil Microbiology

1.3.4 Soil Light
Figure 1.10 highlights the interconversion of energy in the soil–plant system. The key driver for this
interconversion is solar energy. Section 1.3.3 explained how solar energy determines soil tempera-
ture, which in turn influences soil microbial activity. Solar light also directly affects the microbial
distribution and activity at or near the soil surface (where light can penetrate), providing energy
(5% of the net solar radiation drives the photosynthetic activity of plants and microbes) and driving
photoautotrophic soil microorganisms such as certain algae and cyanobacteria. It is generally only
under the conditions of high light and moisture levels that such photoautotrophic soil microorgan-
isms contribute significantly to soil microbial biomass and microbial energy flow through the soil.
Furthermore, the contribution is often short-lived, as soils may dry out or become shaded. Under
the conditions of high photoautotrophic activity, however, considerable amounts of polysaccharides
can be produced in the soil, which play important roles in soil aggregate genesis and stabilization,
thus enhancing the stability of the soil.
The most important effect of light, from a soil microbiological point of view, is that it stimulates
plant seed germination, seedling establishment, and growth and is necessary for some algae and
cyanobacteria to grow on surface soil soils and other surfaces. Plant roots penetrate and aerate
the soil, redistribute the soil water and nutrients through hydraulic lift, and then provide the main
source of carbon substrates for microbial growth. At low light intensity, root metabolic activity and
respiration can be reduced by as much as 50% (Lambers 1987), impacting microbial activity, as
carbon substrates will become limiting (Broughton and Gross 2000).

1.3.5 Soil Atmosphere and Redox Potential

The typical concentrations and diffusion coefficients of the major soil gases in the air and soil atmo-
sphere are indicated in Table 1.3. Because soil water potential (matric and osmotic) is so critical to
determining the extent and nature of soil microbial activity, as well as the diffusive supply of oxygen
to the sites of aerobic activity (contrast the diffusion coefficient of oxygen in air and water), the char-
acteristics of the soil atmosphere are strongly related to the soil water regime. In addition, tempera-
ture is also critical as a rate controller of microbial activity, assuming sufficient water is available.
In relation to the model soil aggregate shown in Figure 1.1, when the aggregate pore space is
water-filled, the aggregate center will first become anaerobic when the demand for oxygen from

Light energy

SOIL PHOTOTROPHS
(plants, algae)

Fixed C
CO2
(chemical energy O2
(H20) from decomposition)

SOIL HETEROTROPHS
(microbes, animals)

Energy lost

FIGURE 1.10 Interconversion of carbon and energy in the soil–plant system. Energy input is in the form of
solar energy and output in the form of heat loss.
The Soil Environment 15

TABLE 1.3
Typical Composition of the Soil Atmosphere Compared to the Open Atmosphere
N2 O2 CO2
Typical concentration in air (%) 79 21 0.035
Typical concentration in soil atmosphere 79 20–21 0.1–1.0
Diffusion coefficient in air (cm2 s−1) 2.1 2.1 1.6
Diffusion coefficient in water (cm2 s−1) 1.6 × 10−4 1.8 × 10−4 1.8 × 10−4
Solubility in water (cm3 L−1) 1.5 1.5 87.8

Diffusion coefficients and solubilities of the main gases are also included.

aerobic respiration cannot be met by the diffusive resupply from the surface of the aggregate. The
central zone of anaerobiosis will grow outward until diffusive resupply is adequate to meet that
demand (oxygen demand from roots and microbes can exceed 20 g m−2 d−1 when activity is high;
Russell 1973). In early work, Greenwood (1975) pioneered our understanding of this microsite onset
of anaerobiosis in water-saturated aggregates, using a derivative of Fick’s law (governing the rates
of gas diffusion) to calculate the critical aggregate radius at which a water-saturated aggregate, with
an active microbial community, generates an anaerobic center:

a 2 = 6CD R

where
a is the critical aggregate radius
C is the distance in O2 concentration between the aggregate surface and the center (mL O2 mL−1)
D is the diffusion coefficient of O2 in water (cm2 s−1)
R is the respiration rate inside the aggregate (mL O2 cm−3)

From Greenwood’s equation, anaerobic conditions are expected to occur in water-saturated aggre-
gates of 1 cm radius or greater for typical levels of soil respiration. From this, we can expect anaero-
bic microsites to develop reasonably often in soil, particularly when there is good aggregation and
recent rainfall and/or poor drainage. In a completely water-saturated soil, more widespread anaero-
bic conditions will generally prevail if this system is biologically active. However, water-saturated
soils are not always anaerobic. Flushed peat soils are often maintained in the aerobic state, with a
continuous resupply of oxygen in the water that flushes through the soil.
Once oxygen is depleted by respirational demand from soil organisms and anaerobic conditions
develop in soil, either in microsites or more widely, the availability of alternative electron accep-
tors determines the (anaerobic) processes that operate (Figure 1.10). These are entirely microbial
processes, as plants and soil animals are obligate aerobes (i.e., they cannot function in the absence
of oxygen). There is a sequence of reduction of terminal electron acceptors, and this is illustrated in
Figure 1.11 along with the prevailing redox potential. The latter is simply a measure of the tendency
of a substance to lose (oxidation) or gain (reduction) electrons and is described by the Nernst equa-
tion shown as follows:

Eh = Eo + 0.059/n ⋅ log ( Ox/Red )

where
Eh is the measured (platinum electrode) redox potential (mV)
Eo is the standard potential of the system (mV)
16 Modern Soil Microbiology

n is the number of electrons in the system

Ox is the number of electrons lost
Red is the number of electrons gained

Understanding the redox chemistry of soils requires a consideration of available electron acceptors
and an awareness of the general rule that the presence of an electron acceptor with a higher oxida-
tion state will inhibit the operation of an acceptor with a lower oxidation state. So, sulfate reduction,
for example, will be inhibited by the presence not only of oxygen but also of nitrate, manganese as
Mn4+, and iron as Fe3+ (Figure 1.11).
The spatial heterogeneity of soils in terms of structure, reducing power (primarily driven by
available carbon supply such as from plant roots or organic residues of plants and animals), and
availability of electron acceptors (oxygen tends to exhibit a gradient of concentrations from the sur-
face to the edge of the rooting depth, and nitrate levels tend to reflect the localized zones of miner-
alization and nitrification) means that redox chemistry is also highly heterogeneous, demonstrating
strong spatial as well as temporal variability.

1.3.6 Soil pH
The soil pH is the negative logarithm of the level of protons per unit volume:

(
pH = log 1  H +  )
t
uc
od

4 +
O n 3+
fin ron

e - e 2+
pr

2S
e - NH

4
t

H
al

H
2
ec

M
e- A
F
20

C
or el

H
pt al

e-

+ e-

e-
c e in

+
ac e rm

e-

SO +
e
M +

4 2-

+
+
3 -

-
O +

3+
2 +

n4

2
T

O
Fe
O

C
M

800
Dissimilatory NO3– reduction
Redox potential (mV)

600
400
Methanogenesis

200
SO42 – reduction
Denitrification

Fermentation
Mn reduction

Fe reduction

0
Respiration

- 200

- 400

0 5 10 15 20 25 30
increasing C(reducing power) arbitrary units
Obligate aerobism

Facultaive anerobism

Obligate anaerobism

FIGURE 1.11 Main redox couples (terminal electron acceptor and redox product) and associated microbial
processes operating in the soil system in relation to redox potential at neutral pH.
The Soil Environment 17

Soil pH is a major determinant of soil microbial distribution and activity (Rousk et al. 2010). The
pH of a soil, or of a microsite in the soil, is the product of numerous factors and processes. First, it
is determined by the parent material from which the soil is formed (acid soils tending to form from
rocks such as granite and alkaline soils from chalks and limestones), as well as the degree to which
mineral weathering has occurred since soil formation. Superimposed on this, a range of biological
processes can modify the soil pH to varying degrees. Figure 1.12 illustrates these pH-modifying
processes, occurring at a range of scales from the microsite (microbial habitat) to the entire bulk
soil. Some of these processes are microbial, and others are animal based, whereas yet others are
driven by the plant. These processes act to create a mosaic of variable pH conditions in the soil.
Consequently, different microbial groups with physiological preferences for particular pH condi-
tions (acidophiles, preferring low pH conditions; alkaliphiles, preferring high pH conditions), as
indicated in Figure 1.12, are favored in soils differing in pH conditions.
Some microorganisms can also thrive in soils with low or high pH conditions (White et al. 1994)
(see Box 1.1).
The distribution and activity of soil microbes as a result of pH is not simply determined by
physiological pH preferences. Many soil microorganisms can tolerate particular pH conditions
that are far from their optimum. For example, many of the fungi found in acid forest soils are
not acidophiles (Killham 1994). When isolated from these environments, some fungi show a
preference for near-neutral conditions, but they are highly competitive under considerable acid-
ity (pH 3 is not uncommon in organic soils, for instance under conifers). A further complication
in understanding pH-related microbial activity in soils is that most microbes are attached to soil

FIGURE 1.12 pH preference of soil microorganisms and the main pH-modifying processes which generate
microhabitats with distinct pH conditions. Explanation: OM = organic matter; OA = organic acids.
18 Modern Soil Microbiology

surfaces and many grow in colonies and even biofilms, which can protect them from the pH pre-
vailing in the soil solution. Growth in biofilms is further discussed in Chapters 4 and 9. We know,
for example, that surface attachment can extend the pH range of chemoautotrophic nitrifiers in
the soil, and we also know that protons can only diffuse slowly through extracellular polysac-
charidic material that binds cells in microbial biofilms. Perhaps for these reasons, and because
many processes in soil are carried out by microorganisms with different pH requirements and/
or tolerance, the soil microbiologist should be wary about any considerations of pH exclusion of
particular processes in soil. Again, using nitrification as an example, for a long time researchers
believed that nitrification would not occur in soils with pH below 5. This assumption came from
in vitro physiological evidence of the pH requirements of the relevant nitrifying bacteria, but it
did not take into account the properties of surface-colonizing populations or of the heterotrophic
nitrifiers, including certain fungi, which have wider pH ranges than their autotrophic counter-
parts (Killham 1994).

1.4 CONCLUDING REMARKS
This chapter examined how soil presents a dynamic and varied habitat, enabling the microorgan-
isms present to interact with each other as well as with plants, animals, and the soil (in its solid, liq-
uid, and gaseous phases) itself. Although, by necessity, key physical and chemical properties of soil
have been considered separately, many of these properties are highly interactive (e.g., temperature
and water), and they act in concert to determine the nature of the soil habitat and, consequently, the
nature of the soil–organism interactions.
We further emphasize that variation in soil properties operates at an impressive range of scales.
This is a vital issue, as variation down to the micrometer scale is relevant to organisms with microm-
eter dimensions, such as bacteria and many fungi. For example, variations in redox potential across
the microporous structure of a soil aggregate ensure that facultatively anaerobic denitrifying bacte-
ria function less than a millimeter away from obligately aerobic nitrifying bacteria that occur nearer
the aggregate surface. The nitrate used in the anaerobic microsites of denitrification is coming from
the aerobic zone of nitrification. Hence, both processes are able to operate simultaneously in close
vicinity despite their mutually exclusive requirements.
The effect of soil on microbes should not be considered a “one-way street.” To the contrary,
the activity of microorganisms is continually shaping the soil habitat itself. For example, much
of the polysaccharides and filamentous materials that hold soil particles together is of ­m icrobial
origin. Also, microbial activity itself can strongly modify soil pH in microsites. In particular,
in the rhizosphere, these are crucial features affecting the life of the microbial inhabitants (see
Chapter 10).

REFERENCES
Bird, N.R.A. and E.M.A. Perrier. 2003. The pore-solid fractal model of soil density scaling. Eur J Soil Sci
54, 467–476.
Broughton, L.C. and K.L. Gross. 2000. Patterns of diversity in plant and soil microbial communities along a
productivity gradient in a Michigan old-field. Oecologia 125, 420–427.
Cha, C., Gao, P., Chen, Y.-C., Shaw, P.D. and S.K. Farrand. 1998. Production of acyl-homoserine lactone
quorum-sensing signals by Gram-negative plant-associated bacteria. Molec Plant Microb Interact
11, 1119–1129.
Elasri, M., Delorme, S., Lemanceau, P., et al. 2001. Acyl-homoserine lactone production is more common
among plant-associated Pseudomonas spp. than among soil-borne Pseudomonas spp. Appl Environ
Microbiol 67, 1198–1209.
Gammack, S.M., Patterson, E., Kemp, J.S., Cresser, M.S. and K. Killham. 1991. Factors affecting the move-
ment of microorganisms in soils. In: Soil Biochemistry, (eds.) J.M. Bollag and G. Stotzky, vol. 7, Marcel
Dekker, New York, pp. 263–305.
The Soil Environment 19

Greenwood, D.J. 1975. Soil physical conditions and crop production. MAFF Bull 29, 261–272.
Killham, K. 1994. Soil Ecology, Cambridge University Press, Cambridge.
Lambers, H. 1987. Growth, respiration, exudation in symbiotic associations: the fate of carbon translocated
to the roots. In: Root Development and Function, (eds.) P.J. Gregory, J.V. Lake and D. Rose, Cambridge
University Press, Cambridge, pp. 125–146.
Marshall, T.J. and J.W. Holmes 1996. Soil Physics, 2nd edition, Cambridge University Press, Cambridge,
pp. 49–53.
Meharg, A.A. and K. Killham. 1989. Distribution of assimilated carbon within the plant and rhizosphere of
Lolium perenne: influence of temperature. Soil Biol Biochem 21, 487–489.
Millan, H., Gonzalez-Posada, M., Aguilar, M., Dominguez, J. and L. Cespedes. 2003. On the fractal scaling
of soil data. Particle-size distributions. Geoderma 117, 117–128.
Murr, L.E., Torma, A.E., Brierley, J.A. (eds.). 1978. Metallurgical Applications of Bacterial Leaching and
Related Microbiological Phenomena, Academic Press, New York.
Paul, E.A. and F.E. Clark. 1996. Soil Microbiology and Biochemistry, Academic Press, New York.
Perrier, E.M.A. and N.R.A. Bird. 2002. Modelling soil fragmentation: the pore solid fractal approach. Soil
Tillage Res 64, 91–99.
Pierson, E.A., Wood, D.W., Cannon, J.A., Blachere, F.M. and L.S. Pierson III. 1998. Interpopulation signaling
via N-acyl-homoserine lactones among bacteria in the wheat rhizosphere. Molec Plant Microb Interact
11, 1078–1084.
Rousk, J., Baath, E., Brookes, P.C. et al. 2010. Soil bacterial and fungal communities across a pH gradient in
an arable soil. ISME J 4, 1340–1351.
Russell, E.W. 1973. Soil Conditions and Plant Growth, 10th edition, Longman, London.
Smith, K. and C.E. Mullins (eds.). 2000. Soil Analysis: Physical Methods, 2nd edition, Marcel Dekker, London.
Standing, D. and K. Killham. 2007. The soil environment. In: Modern Soil Microbiology II, (eds.) J.D. van
Elsas, J.K. Jansson and J.T. Trevors, CRC Press, New York, pp. 1–22.
Vargas, R. and T. Hattori. 1990. The distribution of protozoa among soil aggregates. FEMS Microbiol Ecol
74, 73–78.
White, D., Fitzpatrick, E.A. and K. Killham. 1994. Use of resin impregnated soil sections to assess spatial
location of bacterial inocula introduced into soil. Geoderma 63, 245–254.
Whiteley, M., Diggle, S.P. and P. Greenberg. 2017. Progress in and promise of bacterial quorum sensing
research. Nature 551, 313–320.
Yang, P. and J.D. van Elsas. 2018. Mechanisms and ecological implications of the movement of bacteria in soil.
Appl Soil Ecol 129, 112–120.
 2 The Seven Grand Questions
on Soil Microbiology (Selman
A. Waksman, Reexamined
by Arthur D. McLaren)
Jan Dirk van Elsas
University of Groningen

Paolo Nannipieri
University of Firenze

CONTENTS
2.1 I ntroduction............................................................................................................................. 21
2.2 Developments Examined by McLaren.................................................................................... 22
2.3 What Organisms Are Active under Field Conditions and in What Ways?..............................24
2.3.1 Intracellular Activities in Soil Microbiomes...............................................................24
2.3.2 Activities of Extracellular Proteins in Soil..................................................................25
2.4 W hat Associative and Antagonistic Influences Exist among Soil Microflora
and Fauna?..................................................................................................................... 25
2.5 What Relationships Exist between SOM Transformations and Soil Fertility?.......................26
2.5.1 The Importance of SOM..............................................................................................26
2.5.2 The Concept of Soil Quality........................................................................................ 27
2.6 What Is the Meaning and Significance of the Energy Balance in Soil, in Particular
with Reference to C and N?..................................................................................................... 27
2.6.1 The Processes That Determine the Role of C in Soil.................................................. 27
2.6.2 The Processes That Determine the Role of N in Soil..................................................28
2.6.3 The Flow of Energy in Soil.........................................................................................28
2.7 How Do Cultivated Plants Influence Soil Transformations?................................................... 29
2.8 How Can One Modify Soil Populations and to What Ends?................................................... 29
2.9 What Interrelationships Exist between Physicochemical Conditions in Soil and
Microbial Activities?............................................................................................................... 30
2.10 Conclusions and Perspectives.................................................................................................. 31
References......................................................................................................................................... 33

2.1 INTRODUCTION
Microbiology has flourished ever since the discovery and description of microorganisms as
agents—invisible to the naked eye—that are ubiquitous and have key capacities of transforming
matter and causing disease. The microbiology of soil has been a key area of study, as microbes drive
most of the relevant biogeochemical processes that occur in soil, and immense diversities of micro-
bial functions are present. World-famous basic microbiology courses, first given at Delft University
of Technology (Beijerinck and disciples), and later taken to and developed in the United States by

21
22 Modern Soil Microbiology

Stanier, Phaff, and colleagues, have taken advantage of the immense microbial functional diversity
that can be found in most soils. Thus, a huge diversity of microbial functions has been uncovered
by the use of enrichments from soil, in which conditions were tuned to yield the targeted functional
microbes. In this way, a plethora of nitrogen fixers, ammonia oxidizers, nitrate reducers, sulfate
reducers, iron reducers, next to fermenters, and straightforward aerobic organic carbon degraders
could be isolated from soil. Hence, soil has been depicted, for a long time, as an extremely rich
source of functionally diverse microorganisms.
However, what has remained relatively unknown in the initial stages of the development of
­knowledge in soil microbiology was how the uncovered microbial processes actually “worked”
in situ in the soil. Thus, although the detection of a functional activity following an enrichment may
be taken as evidence for the contention that such a process is actually operational in soil, i­ nformation
about its scale, extent, and timing has been lacking. In his seminal book entitled Principle of Soil
Microbiology (Williams & Wilkins, Baltimore, 1927), Selman A. Waksman described the seven
grand outstanding questions about the living soil (reworded after McLaren 1977), which are as
follows:

1. What organisms are active under field conditions and in what ways?
2. What associative and antagonistic influences exist among soil microflora and fauna?
3. What relationships exist between soil organic matter (SOM) transformations and soil
fertility?
4. What is the meaning and significance of energy balance in soil, in particular with reference
to C and N?
5. How do cultivated plants influence soil transformations?
6. How can one modify soil populations and to what ends?
7. What interrelationships exist between physicochemical conditions in soil and microbial
activities?

As we will learn in this book, the living soil stands out as the most organism- and function-diverse
habitat that exists on this planet. Moreover, the spatial and temporal intricacies encountered in
most soils pose specific challenges to our understanding of the local processes that take place
(see Chapter 1). Hence, when reconsidering the aforementioned seven grand questions that were
posed almost 100 years ago, one may safely state that most of these still constitute the challenges
in soil ­microbiology research of today. In an editorial in Soil Biology and Biochemistry written
50 years after Selman Waksman published his seven grand questions, Arthur Douglas McLaren
(1977), the “father” of soil biochemistry, reexamined these questions. In this chapter, we discuss to
what extent progress has been made with respect to each of the Waksman questions, as reexamined
by McLaren (Figure 2.1).

2.2 DEVELOPMENTS EXAMINED BY McLAREN

McLaren (1977) briefly summarized the advances in the body of knowledge on the seven ­questions
that emerged since the publication of the questions by Selman Waksman. These advances ­concerned
the interactions between microbes and plants in soil, the distribution of microorganisms in the
soil matrix, the ways to quantify nutrient transformations in soil (by using labeled compounds),
and the possibility to set up quantitative mathematical models simulating nutrient dynamics in
the soil–plant system. Moreover, the discovery of beneficial bacteria stimulating plant growth by
­producing ­phytohormones, the preferential growth of bacteria on some parts of the roots, the role
of ­bacteria in the biological control of plant disease, and the role of mycorrhizae and other benefi-
cial ­microorganisms in plant nutrition were reviewed. The first experiment on nutrient dynamics
with labeled compounds was carried out by Norman and Werkman (1943), who showed that 20%
of 15N-enriched soybean residues is taken up by plant roots, whereas most of the remainder is
The Seven Grand Questions 23

FIGURE 2.1 Depiction of the seven grand questions of Selman A. Waksman in soil (microbiome) functioning.

incorporated into the SOM. Later, Jenkinson and Powlson (1976) set up a method for d­ etermining
the size of the soil microbiome, which enables to trace the dynamics of nutrients through soil
microbial communities. This constituted an important advance in the quantification of nutrient
behavior in the soil–plant system and in improving models simulating this dynamics (Table 2.1).
Major developments have taken place, with respect to all seven questions, in the decades that
passed since these early achievements. Most of the developments have been driven by strong meth-
odological advances in soil microbiology, as testified in various other chapters in this book (see,
e.g., Chapters 12–19). Here, we briefly revisit each of the seven grand questions on our current
understanding of the living soil.

TABLE 2.1
The Seven Grand Questions of Waksmann and Progress Made To Date
References/
Specific Question Progress Made Future Prospects Remarks
What organisms are Novel methods have slowly allowed Soil activity is site- and Chapters 15
active under field overall and targeted analyses of condition-specific, and so targeted and 16
conditions and in what activity transcriptomics/proteomics
ways? approaches are required
What associative and Key rhizosphere inhabitants and their Targeted studies using selected Chapters 9,
antagonistic influences potential roles are understood. The novel methods, including 21, and 22
exist among soil complexity of the rhizosphere still meta-omics, are needed
microflora and fauna? poses a big hurdle
What relationships exist SOM is a key determinant of soil Increasing the SOM content to Garcia et al
between SOM fertility. Soil fertility is now also counteract the release of CO2 to (2018);
transformations and soil denominated soil quality the atmosphere Chapter 20
fertility?
What is the meaning and Growth of heterotrophic To distinguish soil Schimel and
significance of energy microorganisms decreases when microenvironments according to Bennett
balance in soil, in available C and/or N are low their C/N availability (2004)
particular with reference
to C and N?
(Continued)
24 Modern Soil Microbiology

TABLE 2.1 (Continued )

The Seven Grand Questions of Waksmann and Progress Made To Date
References/
Specific Question Progress Made Future Prospects Remarks
How do cultivated plants Rhizospheres and mycorrhizospheres To simulate interactions between Chapter 10
influence soil are now known as major “changers” plants, microorganisms, and fauna
transformations? of local activities in microcosms
How can one modify soil Many studies on soil inoculants have Future endeavors should attempt to Chapter 22
populations and to what revealed the microbiostatic character create specific niches for
ends? of soil. Soil management, for (incoming) beneficials in soil, for
example, by adding substrates, is a example, by adding nutrients or
promising avenue specific microhabitats
What interrelationships Key factors have been studied. Soil Determination of these effects at the Chapters 1
exist between water, pH, and SOM are the key microscale level will allow better and 2
physicochemical determinants of activities insights
conditions in soil and
microbial activities?

2.3 WHAT ORGANISMS ARE ACTIVE UNDER FIELD

CONDITIONS AND IN WHAT WAYS?
2.3.1 Intracellular Activities in Soil Microbiomes
Questions about microbial activities in soil are broad and needs to be defined in the ­context of
the research aims as well as the specific conditions. For instance, the question may pertain to
the activity of specific ammonia oxidizers in soil under aerobic versus anaerobic c­ onditions.
Alternatively, it may be broader, addressing the aerobic degradation of lignocellulose ­material
in the soil. Answers to each of these issues will require particular approaches and t­ echniques,
which are often molecularly based (see Chapters 12–17). However, there are still u­ nresolved
­methodological problems that hamper progress in this area. Current advanced ­techniques to study
soil ­m icrobiomes are based on analyses of soil DNA, RNA, and/or proteins. In addition, methods
that allow to track and trace the fate of marker atoms (in molecules), for example, 13C, 14C, and
15N, have enabled to reveal the efficiencies of key processes in soil, such as photosynthesis and

translocation of ­photosynthate to belowground organisms (Drigo et al. 2009), organic carbon

breakdown, nitrogen fixation, ­a mmonia oxidation, and nitrate reduction. A key method here is
stable isotope probing, in which the incorporation of stable label, for example, 13C, into biomole-
cules (often DNA), is ­measured. See Chapter 17 for details. A combination of the aforementioned
methods is recommended, as each offers a d­ ifferent perspective on (potential) function, with
discrepancies being related to molecule stability (and thus timing of events). Also, the classical
methods have shown puzzling results. For example, transmission electron microscopy of soil
sections has shown that bacterial cells are mainly present in connection to plant residues in soil,
often occurring in the interior of these debris (Foster et al. 1983). Supportive of the key presence
of microbial cells within such refuges was the fact that chloroform fumigation primarily lyses
cells of bacteria present between aggregates but not those of bacteria inside aggregates (Ladd
et al. 1996). Unfortunately, the application of ultra-cytochemical activity tests did not detect
active enzymes (and by inference active microbes) in soil due to the presence of cross-reacting
electron-dense soil minerals and/or SOM (Ladd et al. 1996).
Research on the activity of microbes in soil also needs to consider the spatial and temporal
aspects of soil, as these continuously influence the local cellular activities. It is possible that,
The Seven Grand Questions 25

TABLE 2.2
Methodological Progress That Has Driven the Advancement in Our Knowledge about the
Seven Grand Questions of Selman A. Waksman
Remarks/
Advanced Method What Has It Brought Us? References
Use of tracers/label for Broad view of fate of labeled compound, for example, C and N Nannipieri and
compounds compounds Paul (2009)
Use of reporter genes/biosensors Very sensitive in situ detection of specific microbial activities, Van Overbeek and
including responses to soil conditions van Elsas (1995)
Soil metagenomics Overall vision of soil phylogenetic and functional diversity Chapter 14
Soil metatranscriptomics Specific vision of expressed genes at messenger RNA (mRNA) Chapter 15
level
Soil metaproteomics Specifically expressed proteins Chapter 16
Stable isotope probing Enables monitoring an actual process in soil, for example, Radajewski et al.
methane/methanol oxidation (2002),
Chapter 17

given the local nature of “conditions,” a microbial population spread through the soil can show
high activity levels at particular sites and/or times, while being virtually silent at o­ thers. Activity
measurements, either considering output parameters such as released CO2 or fixed N2, or directly
measuring expressed genes, will inevitably yield data that are averaged over a ­certain volume of
soil. Such data do not precisely reflect the underlying plethora of local activities (Table 2.2).

2.3.2 Activities of Extracellular Proteins in Soil

A major characteristic of soil as a biological system is the capacity of surface-reactive soil
­particles to adsorb key biological molecules, such as proteins (and thus enzymes) and nucleic acids
(Nannipieri et al. 2003). McLaren and Peterson (1967) reported that the adsorption of enzymes to
the surface-reactive particles in soil protects them against proteolysis without activity losses. As
discussed in Chapter 16, the extracellular protein- and peptide-N, stabilized by surface-reactive
soil particles or entrapped in soil aggregates, may make up 30%–50% of the soil organic N. This
is higher than the organic N (mostly protein-N) that is intracellularly localized (4% on a­ verage) in
the soil matrix. The bibliography on stabilized extracellular proteins in soil is extensive (Nannipieri
et al. 2012) and exceeds that related to extracellular DNA in soil (Pietramellara et al. 2009).
Generally, a focus has been placed on extracellular stabilized hydrolases (such as urease, proteases,
and ­phosphomonoesterases), and it was found that their stabilization depends on the adsorption onto
surface-reactive particles (such as clays) or entrapment in the organo-mineral complexes. These
enzymes can persist in soil as long as the aggregates or organo-mineral complexes entrapping them
are not broken (Nannipieri et al. 2012).

2.4 WHAT ASSOCIATIVE AND ANTAGONISTIC INFLUENCES

EXIST AMONG SOIL MICROFLORA AND FAUNA?
Since the incipience of the concept of microbial (associative and antagonistic) interactions in soil,
great progress has been made in this area. This pertains to both the interactions between d­ ifferent
members of soil microbiomes and those between these and components of the soil fauna and/or
the roots of plants. In classical cultivation-based work, a large range of either antagonistic or syn-
ergistic interactions among soil microorganisms has been identified. Chapter 9 discusses some of
26 Modern Soil Microbiology

the interactions that occur between the different soil microbes. It also places a focus on the way
microbes occur in the soil (spatially explicit, often in biofilms adhering to surfaces). It is exactly
here that the crux with respect to our understanding of the soil microbiome lies. Clearly, the het-
erogeneous and complex spatial structure that occurs in most soils has a strong bearing on the
interactions, which are most often spatially explicit. Thus, it is precisely in this area that most of the
progress has been and will continue to be made.
A particular case in point is found in the so-called hot spots for activity in soil, such as the
­rhizosphere, the mycosphere, and other such “spheres.” In these habitats, we see a range of
­developments of our knowledge. In some cases, microbe–microbe interactions are understood
to the finest molecular level, and such data have mostly come forward in studies performed in
soil-mimicking systems. For instance, in a study performed on soil extract agar, Haq et al. (2017)
found a five-gene cluster that is involved in energy generation (presumably at the expense of
­f ungal-exuded glycerol and oxalate), to be highly upregulated in Burkholderia (recently reclas-
sified to Paraburkholderia) terrae in interaction with the soil fungus Lyophyllum sp. strain
Karsten. The gene cluster apparently endowed the organism with a “kick-start” machinery that
enabled it to immediately respond to emerging fungal exudate. A large suite of other current
developments with respect to the mechanisms that underlie interactions in soil also aims to
unravel the molecular events that trigger particular associative or antagonistic responses (see
Chapter 9). What emerges from the large bibliography on this topic is that, over evolutionary
time, soil has acted as a complex and diverse matrix, in which a huge diversity of symbiotic,
neutral, and/or antagonistic interactions has emerged, as driven by the Darwinian rules of evo-
lution. However, we still are far from understanding how soil modulates such interactions, and
so it is foreseen that, next to studies in soil-mimicking systems, real-world soil studies remain
necessary in future research efforts.

2.5 WHAT RELATIONSHIPS EXIST BETWEEN SOM

TRANSFORMATIONS AND SOIL FERTILITY?
2.5.1 The Importance of SOM
Already in early work, it became apparent that soil fertility was intrinsically linked to the organic
matter in the soil, notwithstanding the existence of some exceptions to this rule. The exception
illustrated by a curious finding described by McLaren (1977). They found an allophanic soil to
have low fertility despite its high organic C content. A decrease in the mineralization processes
(and thus of the release of nutrients that could be taken up by the plant) was found in this soil,
and organic compounds turned out to be complexed with allophanes. In current research, the
use of labeled organic compounds (see Section 2.3) allows us to determine the quantitative
dynamics of nutrients in the soil–plant system. For example, the use of 15N-enriched fertilizer
can ­d istinguish between the behavior of fertilizer N and that of soil N, and thus quantify what
percentage of plant N uptake is satisfied by the added fertilizer compared to that of the N min-
eralized from soil organic N. The so-called priming effect, that is, the change in the mineraliza-
tion rate of SOM due to the addition of an organic compound to soil, can be determined by using
C-labeled compounds.
The role of SOM as the basis of soil fertility across many soils has thus been extensively
­documented. However, human activities over the past decades have significantly changed the regional
and global balances of SOM, with effects on climate change. This has often been d­ etrimental to soil
fertility (Garcia et al. 2018). Moreover, there is a need to preserve or even enhance SOM in soils
given the negative effects of SOM depletion on the climate. Therefore, both agricultural and forest
management practices are increasingly aiming to store organic C in soil to counteract the release of
CO2 into the atmosphere (see also Chapter 25). In addition, interactions between SOM and complex
human–natural systems require new research into regional and global SOM budgets.
The Seven Grand Questions 27

2.5.2 The Concept of Soil Quality

In 1994, Doran and Parkin introduced the concept of soil quality, defined as “the capacity of a soil
to function within ecosystem boundaries to sustain biological productivity, maintain e­ nvironmental
quality and promote plant and animal health.” This definition is broader than that of soil fertil-
ity since it also contains the role of soil in plant and biological productivity and in attenuating
the impact of environmental contaminants and pathogens. In addition, this definition addresses
the importance of soil for plant, animal, and human health. It is important to determine changes
in soil quality, since soils are affected by climatic changes as well as human activities, ­including
intensive agricultural practices. Soil quality is related to the biological, chemical, and physical
­properties of soil, but unfortunately these parameters are interwoven and it is not possible to have
an ­unequivocal evaluation of (changes in) soil quality. In a recent paper (Schloter et al. 2018), the
need was e­ mphasized to develop robust, reliable, and resilient molecular indicators for monitoring
soil quality. Besides, biological, chemical, and physical indicators have been selected. However,
the chemical and physical properties of soil are less sensitive than the biological ones, and this
has a bearing on (subsequent) changes in SOM content, which is related to soil quality (Nannipieri
et al. 2003). In addition, biological indicators of soil quality have, in the past, mainly focused on
the visible part of the soil biota, neglecting the microbiome (Schloter et al. 2018). Hence, the need
to develop indicators reflecting soil microbial activity has been stressed, as soil microbiomes play
predominant roles in affecting soil functionality (Schloter et al. 2018, Stott et al. 2009). However, of
the ten indicators that have been proposed for evaluating soil quality, only microbial biomass C and
potential nitrification were based on microbiological parameters (Andrews et al. 2004). Therefore,
Schloter et al. (2018) proposed to take into consideration genes encoding proteins that are involved
in key microbial processes, such as several steps of the N, C, and P cycles, in soil. Several such
genes were proposed for development into indicators and others can be derived from the e­ xtensive
bibliography on molecularly based soil analyses.

2.6 WHAT IS THE MEANING AND SIGNIFICANCE OF THE ENERGY

BALANCE IN SOIL, IN PARTICULAR WITH REFERENCE TO C AND N?
2.6.1 The Processes That Determine the Role of C in Soil
Organic C plays a key role in soil functionality, as it affects the biological, chemical, and p­ hysical
properties of soil. The biological properties include soil biodiversity. Soil has a source/sink role
in terms of carbon, so it can act as either a net carbon source or sink. As briefly discussed in
chapter 1, carbon stably localized in the soil—in the SOM—is often present in highly complex
­macromolecules, whereas labile C encompasses smaller molecules that are more prone to utilization
and conversion into biomass and CO2. Current aims for soil (microbiome) management practices are
to foster the organic C storage function of soil in order to counteract an increase in atmospheric CO2
levels (Garcia et al. 2018). This implies that CO2 fixation (autotrophic) processes are to be stimulated
at the expense of C “burning” (heterotrophic) processes. In soil, the former processes are mainly
carried out by plants, algae, and a range of autotrophic bacteria, including cyanobacteria.
The carbon-use efficiency by the soil microbiota has often been studied by a “holistic” approach,
considering the soil microbiome as an entire pool (microbial biomass C) and the C respired as CO2.
Anderson and Domsch (1990) proposed the qCO2 (the ratio between C–CO2 and microbial biomass
C) as an index to evaluate the amount of organic C that is incorporated into microbial biomass.
High qCO2 values were posed to be indicative of microbial stress, since under stress conditions, a
higher amount of C is respired to repair metabolic damages due to the stress. However, the ratio
also depends on factors other than stresses. For example, changes in the fungal/bacterial biomasses
can affect the ratio, since fungi respire less C than bacteria per unit incorporated C (Wardle and
Ghani 1995).
28 Modern Soil Microbiology

2.6.2 The Processes That Determine the Role of N in Soil

The second major element of importance to soil microbiomes is N. Soil N is subjected to cycling
across the different chemical forms, as further outlined in Chapter 11. The N cycle in soil is
characterized by many reactions and losses. Biological N fixation, nitrification, denitrification, N
mineralization and immobilization/turnover, ammonia volatilization, N leaching, and N loss by
runoff are processes that have been known even before the time of Waksman and later McLaren.
In ­addition, even more cycling steps have been discovered later on. For example, N2O can be
produced not only through denitrification but also by nitrifier denitrification (Nannipieri and
Paul 2009). Anaerobic ammonia oxidation (Anammox), the oxidation of ammonia to N2 gas, with
concomitant reduction of nitrite, can occur under anaerobic conditions in soil (Nannipieri and Paul
2009). The oxidation of ammonia to nitrite, the first step of the nitrification process, is carried out
not only by ammonia-oxidizing bacteria but also by ammonia-oxidizing archaea. Also, a novel
finding indicates the existence of a complete nitrification pathway in drinking water purification
sand beds in one organism, Nitrospira, instead of in the classical two-species process carried out
by Nitrosomonas (oxidation of ammonia to nitrite) and Nitrosospira (oxidation of nitrite to nitrate)
(Daims et al. 2015). It is an open question to what extent the two-species consortium ­outcompetes,
and thus prevails over, the single-organism system in soil settings. Finally, ammonia-oxidizing
bacteria such as Nitrosococcus and Nitrosospira species also contain genes catalyzing the enzymes
of the denitrification process (Norton et al. 2008). The N cycling processes are further detailed in
Chapter 11.
Generally, if N is the limiting nutrient, the degradation of organic C will increase as it adds
inorganic N to the soil. Similarly, if P and S are the limiting nutrients in soil, the degradation rate
will increase if inorganic P and/or S compounds are added to soil, respectively. However, this is not
always observed, since the quality of the soil organic C is also an important determinant. For exam-
ple, the degradation of lignin decreases with high N availability, since ligninases that are responsible
for lignin degradation are inhibited by inorganic N (Knorr et al. 2005). According to the ecological
stoichiometry theory, elemental ratios in soil determine nutrient retention and biomass production
(Sterner and Elser 2002). As a consequence, the elemental stoichiometry of soil microbial biomass
(with a mean C:N:P ratio of 60:7:1) will determine the microbial C, N, and P demand in relation
to the availability of these elements in soil (Sinsabaugh et al. 2009). The mean ratio of activities
of four enzymes [β-1,4-glucosidase, β-1,4-N-acetylglucosaminidase, leucine a­ minopeptidase, and
acid or alkaline phosphomonoesterase (the latter measured depending on soil pH)], involved in C,
N, and P dynamics, was about 1:1:1 in all investigated soil and sediment ­samples (Sinsabaugh et al.
2009). However, whereas β-1,4-glucosidase and both phosphomonoesterase activities are proxies
of organic C and organic P mineralization, respectively, there is no evidence that both β-1,4-N-
acetylglucosaminidase and leucine aminopeptidase activities can serve as proxies of organic N
mineralization (Nannipieri et al. 2018).

2.6.3 The Flow of Energy in Soil

Regarding the flow of energy in soil, Gray and Williams (1971) already suggested that most of
the energy inputs into soil (from solar energy captured by plants, algae, and cyanobacteria) will
serve to maintain microbiomes rather than allowing much microbial growth. This has resulted in
the concept of extended microbial generation times in most soils. In other words, the (slow) build-
ing of new microbial biomass is offset by the gradual death of “old” parts of the biomass, and this
results in a somewhat dynamic equilibrium. Both soil conditions and the rate of energy input would
thus determine the biomass dynamics, resulting in the concept of a “carrying capacity,” which is
characteristic for each soil. The underlying average low growth rates in most soil microbiomes
indicate that microbial transformations would be also slow, on average. However, these calculations
have been based on microbial biomass as a whole, without considering that soils show hot spots
The Seven Grand Questions 29

(sites with enhanced microbial densities and activities) and “hot moments” that are characterized
by the (sudden and ephemeral) presence of available substrates for microbial growth (Kuzyakov
and Blagodatskaya 2017). The calculation of microbial growth rates in soil inside and outside of the
hot spots (sites with enhanced microbial densities and activities) and/or hot moments (sudden and
ephemeral) is still a challenging research aim, also considering that soil microorganisms can form
resting structures under starvation conditions.

2.7 HOW DO CULTIVATED PLANTS INFLUENCE SOIL TRANSFORMATIONS?

It is well established that, in most soils, the soil under the influence of the roots (rhizosphere soil)
has a larger microbial biomass with higher activity than the bulk soil. This so-called rhizosphere
effect is due to the release of photosynthates and derived compounds produced by the plant, and to
rhizodeposition of compounds from lysing root epithelial cells. Garbeva et al. (2004) examined the
relative effects of soil type, plant type, and agricultural management on soil microbiomes. Soil
type, as reflected in the soil properties, is often more important than plant species type in affecting
the microbial diversity of rhizosphere soil, at least after a few years of monoculture. De Ridder-
Duine et al. (2005) showed that Carex arenaria, a nonmycorrhizal plant species (chosen to avoid
the confounding effect by different mycorrhizal colonization), cultivated in ten soils with different
properties, did not affect the local bacterial diversities, as shown by polymerase chain reaction
(PCR) followed by denaturing gradient gel electrophoresis. Instead, these depended on soil proper-
ties. Inceoğlu et al. (2010) revealed subtle differences in rhizosphere bacteriomes (assessed by DNA
sequencing) exerted by the roots of different potato genotypes in the same soil. Hence, the effects
are diverse and depend strongly on the system used as well as the methods of analysis. Moreover,
these studies did not address the effects on microbial functions. With respect to these, a recent
study showed that maize with a higher N-use efficiency (NUE) selected bacterial communities in
the rhizosphere soil differing in β-glucosidase-encoding genes and with a higher β-glucosidase
activity than maize with lower NUE (Pathan et al. 2015). Moreover, the former line also had higher
protease activity and higher abundance of protease-encoding genes in its rhizosphere than the lat-
ter. It is important to underline that spatiotemporal effects are important in the rhizosphere, which
can be studied under laboratory conditions, using rhizoboxes (Neuman and Romheld 2007). For
example, root exudation is mainly confined to apical root zones and is not always constant but fol-
lows diurnal rhythms. A metatranscriptomics study showed the activities of many prokaryotic taxa
were increased by root exudation from barley plants at the pre-dawn compared to the post-dawn
stage (Baraniya et al. 2017). Therefore, it is plausible to hypothesize that, under field conditions, the
rhizosphere effect comes in waves in dependency of sunlight, whereas it is lost when soil cropped
to monoculture is plowed after harvest.

2.8 HOW CAN ONE MODIFY SOIL POPULATIONS AND TO WHAT ENDS?

The importance of soil type, reflected in soil properties, as a driver of the composition of soil
­microbiomes, was first shown, across a suite of soils, by Gelsomino et al. (1999) and later c­ onfirmed
by several other studies. For instance, Delmont et al. (2014) recently studied the microbial diversity
of two soils (one from a grassland of the long-term experimental site at the Rothamsted Experimental
Station in Harpenden, England, and the other one from a forest in Vallombrosa, Firenze, Italy) treated
to have either the same microbial community or that from the other soil. The study showed that the
functional and taxonomic composition of the respective microbiomes was that of the soil receiving
the inoculum and not that of the “donor” soil. Hence, soils may be rather “recalcitrant” to microbi-
ome changes, with a presumed large effect of the soil matrix. Presumably, the type of soil matrix
determines, to a large extent, the distribution of nutrients, water, and other “conditions” over the soil
aggregates and pores. It is therefore the key driver of the distribution of habitable niche space for
adapted microbial forms, resulting in the apparent recalcitrance of soil microbiomes to change.
30 Modern Soil Microbiology

However, for many purposes, such as the biological control of plant diseases, fertility enhance-
ment, and/or bioremediation, it may be desirable to modify soil microbiomes, as already suggested
by Waksman (1927). The aim would be to establish novel organisms that are beneficial to soil
­function, in terms of either fertility or plant health stimulation, or to promote the degradation of, for
instance, polluting compounds. Such aims have been on the research agenda for several decades.
These were also the main themes in the early research of Waksman (1927).
One of the key approaches taken to modify soil microbiomes has been the introduction of (micro)
organisms with the desired function, whereas another approach has relied on the modulation of
soil parameters, for instance, by the addition of “substrates” such as manure, biochar, and/or crop
residues, with the aim to promote the occurrence as well as activity of naturally present beneficial
organisms.
Although there are accounts of successful introductions, for instance, of rhizobia that sup-
port soil nitrogen acquisition from the atmosphere, major obstacles have been identified. These
lie in the recalcitrance of soil to change following organism introductions. As discussed in the
­foregoing, the reason for this “recalcitrance” may to a large extent lie in the lack of an open niche
for the incoming organisms (van Veen et al. 1997, Mallon et al. 2015), a phenomenon denoted as
soil ­microbiostasis. Although we have greatly improved understanding of how plant-­beneficial
organisms may work (see Chapter 22), the success of introduction of many of such o­ rganisms is
not guaranteed as a result of the lack of knowledge as to how to give them an “open niche” in
the soil.
With respect to the second option of modulating the soil microbiome by soil amendments, the
aforementioned review by Garbeva et al. (2004) is important, as it discusses the drivers of soil
microbiome composition, which are as follows:

1. Plant driven
2. Soil type driven
3. Soil management driven

All the three groups of complex drivers are deemed to be important, and with respect to their
effects on soil microbiomes, they were split up into several subfactors: types and levels of nutrients/
organic matter (e.g., from root exudates, soil reshuffling by, for instance, plowing), pH level (e.g., as
affected by soil processes), water regime (e.g., affected by plant water suction, agricultural regime,
soil type), and other factors (e.g., biotic stresses exerted by predators). Although, as we have seen,
soil ­microbiomes are often highly recalcitrant to change, they may be shifted, albeit slowly, when
any of such soil parameters is changed/modulated. It appears the changes that can be effectuated
are highly context-dependent (soil and climate type), and therefore, research is needed on a case-by-
case basis, in order to develop the best practices for changing the activity, biomass, and composition
of soil microbiomes in the planned direction.

2.9 WHAT INTERRELATIONSHIPS EXIST BETWEEN PHYSICOCHEMICAL

CONDITIONS IN SOIL AND MICROBIAL ACTIVITIES?
It is currently known that, among other drivers, particular abiotic soil conditions drive the structure
and activity of soil microbiomes. Here, it is important to discern the extent to which the different
drivers operate, and to what extent they affect the different members of soil microbiomes. As a case
in point, it has been amply shown, across a wide range of soils, that soil pH is an important driver
of the structure of soil bacterial communities, with pH dropping to values below 5.0 being strongly
selective (Rousk et al. 2010). See also Chapter 1 and Box 1.1. This finding is easily connected to
our understanding of the behavior of bacteria under changing pH; a pH value below 5.0 is strongly
prohibitive for most bacteria and allows a distinction of acid-sensitive from acidophilic types. On
the other hand, soil fungi are less affected by changes in soil pH. For example, particular fungi,
The Seven Grand Questions 31

such as Penicillium and Fusarium spp., can grow at pH values of up to 13, whereas other fungi
prefer slightly acidic conditions (around pH 5.2–5.5) (Thorn 2012; see Chapter 5). Fungal activity in
the soil can thus be stimulated by the presence of (acidic) organic compounds of plant and animal
origin. Their role in degrading cellulose, hemicellulose, lignin, and chitin is discussed further in
Chapter 5.
As outlined in Chapter 1, the soil water regime is a strong regulator of soil microbial life. Soil
water is obviously dependent on weather (rain versus drying) conditions, as well as soil drainage
characteristics. Soils with poor drainage characteristics (e.g., clayey soils), when exposed to frequent
heavy rainfall, exhibit prevailing conditions of water saturation, which incite anoxia. This stands
in strong contrast to easily drained soils (e.g., sandy soils), which may turn out to be largely oxic,
on average. However, the latter regime may imply that periods of drought also occur, which may be
extensive. Under such dry conditions, the activities of local microbiomes will be v­ irtually stalled.
Thus, the consequences of a soil’s prevailing water regime, resulting in water levels ­varying between
0% and 100% of the water-holding capacity, and thus oxia versus anoxia, are large. The respective
microbiomes will need to shift their metabolisms from virtually stalled (period of drought) to aero-
bic respiration (oxia in most soil pores) to anaerobic respiration (using alternative terminal electron
acceptors, see Chapter 1) or fermentation (anoxia in most pores).
Finally, agricultural management regime is an important determinant of soil microbiome
­dynamics, as local conditions in the soil can be heavily affected by measures such as plowing,
­tilling, crop rotation and fertilizer and animal waste application. The bibliography on the effects
of ­different agricultural management regimes, causing different environmental effects, on soil
­microbial ­diversity is growing, but the respective findings should only be reviewed when data
of ­different soils and agricultural managements are obtained. One key facet, discussed in many
­scientific fora, is the tendency of microbial diversity to become “eroded” with progressively
enhanced intensity of (­agricultural) land use. This phenomenon apparently has implications for
important soil c­ haracteristics, for example, the suppressiveness to plant diseases (further discussed
in Chapter 21).

2.10 CONCLUSIONS AND PERSPECTIVES

After about 100 years of research in soil microbiology, we can divide the s­ cientific ­developments
into several parts, i.e., the period from the early microbiology era (in which Winogradsky, Beijerinck
and Waksman played pivotal roles), via the era typified by McLaren (soil biochemistry and pro-
cesses) to the period up to today. Figure 2.2. provides a graphical depiction of the development of
soil microbiology, highlighting some important findings. Considering this figure, it comes to our
minds to ask : “where do we stand now with respect to understanding and fostering the functionality
of our soils, and what are the decadal aims for the future?” The availability of a sufficient number
of high-quality and highly fertile soils is at the basis of the sustenance of life on this planet. The
key to survival of humans under the effects of a fast-growing population and the current climate
change lies in our capabilities to preserve and/or improve our precious living soils in terms of their
functioning and their quality. In particular, mitigation of the rising atmospheric CO2 levels requires
increased attention to the potential to enhance the inherent capacity of many soils to act as effective
carbon sinks.
With respect to understanding the living soil and its functions, one may safely posit that enor-
mous data sets have been generated that document the capacities of diverse soil organisms with
respect to the transformation of key biochemical compounds in the soil, the interactions with other
organisms, and the modulation of soil quality and f­ ertility. However, we are still far from a complete
understanding of the functioning of the living soil s­ ystem. It is important to understand that this
function should be considered in the context of the ­particular system studied (context dependency).
In terms of the fate of key compounds, we can quantify the dynamics of at least some nutrients, by
using a holistic approach and with the help of labeled compounds. For example, in the case of N, the
 32

FIGURE 2.2 Time line depicting major developments and key scientists in soil microbiology over about 120 years. *Discussed further in Chapter 3.
Modern Soil Microbiology
The Seven Grand Questions 33

main pools in soil are microbial biomass N, organic N, exchangeable and fixed (or non-exchangeable)
ammonium-N, nitrate-N, and N taken up by plants. In one study, ammonium accounted for almost
25% of 15N-enriched urea applied to a ­sorghum field, whereas the fertilizer taken up by the sorghum
accounted for 21% and that ­immobilized into ­microbial biomass N was about 5% at harvest time
(Nannipieri et al. 1999).
However, our analytical methods are still far from optimal and are not yet sufficiently fine-tuned
for the analysis of a wide range of soil types. This, in combination with the enormous diversity
of organisms in most soils, and the highly intricate network of interactions that can occur, in a
­spatiotemporally explicit manner, across these soils, prevents us from being all-encompassing in
all aspects of soil microbiome analyses. As we learn in Chapters 1 and 3, even within a single soil
the level of homogeneity may be low. Most soils in the world consist of a collection of “islands” for
microbial life and function, where the ecological rules that determine soil function may all have to
be locally determined.
How can we move forward in our analysis and exploration of soil functions? Given the
immense diversity and variation across soils and their microbiomes, we advocate that future
studies should (1) be performed on a case-by-case basis and (2) consider the local heterogeneity
within the soil compartment that is being analyzed. In other words, each soil system needs to be
considered as an intricate system in itself, taking into account its own intricacies and legacies.
It may be too narrow to just compare experimental outcomes in one soil to those in other soils.
Rather, a deeper understanding of each soil system must come from repeated studies of the same
system, using variations of conditions and sampling scale, and including replicated time series
of development. The proposal to define the Rothamsted soil—where over 100 years of historical
study provide an excellent baseline—as a “model” soil for exploration by meta-omics techniques
is a good example of such an approach (Vogel et al. 2009, Delmont et al. 2012). However, in
the first analyses that were performed, only 34.5% of the metagenomics reads were assigned
to function, with only <1% of the annotated sequences fitting already-sequenced genomes at
96% similarity. This indicates that most soil microbiomes are still understudied. Therefore, fur-
ther improvements in technology and further imaginative research efforts are needed to bring
sufficient progress with respect to our ­understanding of the microbiome and its diversity and
activity in soil.

REFERENCES
Anderson, T.H. and K.H. Domsch. 1990. Application of eco-physiological quotients (qCO2 and qD) on
­m icrobial biomass from soils of different cropping histories. Soil Biol Biochem 20, 107–114.
Andrews, S., Karlen, D.L. and C.A. Cambardella. 2004. The soil management assessment framework: a
­quantitative soil quality evaluation method. Soil Sci Soc Am J 68, 1945–1962.
Baraniya, D., Nannipieri, P., Kublik, S. et al. 2017. The impact of the diurnal cycle on the microbial
­transcriptome in the rhizosphere of barley. Microb Ecol 75, 830–833.
Daims, H., Lepedeva, E.V., Pjevac, P. et al. 2015. Complete nitrification in Nitrospira bacteria. Nature
528, 504–509.
De Ridder-Duine, A.S., Kowalchuk, G.A., Klein Gunnewick, P.J.A. et al. 2005. Rhizosphere bacterial
­community composition in natural stands of Carex arenaria (sand Sedge) is determined by bulk soil
community composition. Soil Biol Biochem 37, 349–357.
Delmont, T.O., Prestat, E., Keegan, K.P. et al. 2012. Structure, fluctuation, and magnitude of a natural
­grassland and metagenome. ISME J 6, 1677–1687.
Delmont, T.O., Francioli, D., Jacquesson, S. et al. 2014. Microbial community development and unseen
­diversity recovery in inoculated sterile soil. Biol Fertil Soils 50, 1069–1076.
Doran, J.W. and T.B. Parkin. 1994. Defining and assessing soil quality. In: Doran, J.W., Coleman, D.C.,
Bezdicek, D.F., Stewart, B.A. (Eds). Defining Soil Quality for a Sustainable Environment. Soil Science
Society of America and American Society of Agronomy, Inc., Madison, WI, pp. 3–21.
Drigo, B., van Veen J.A. and G.A. Kowalchuk. 2009. Specific rhizosphere bacterial and fungal groups respond
differently to elevated atmospheric CO2. ISME J 3, 1204–1217.
Exploring the Variety of Random
Documents with Different Content
 The Fairy’s Party

Neighbors passing the house of the little Fairy Health, one lovely
summer day, noticed a most unusual stir and bustle. Large and small
packages were continually arriving and the neat little housemaid was
kept quite busy opening and closing the door to receive them.
The Fairy, flitting here and there among the flower beds, was
accosted by one curious neighbor and asked what it all meant.
With her hands full of flowers the Fairy paused to answer him:
“I am giving a party this afternoon,” she said, “to friends from a
distance, and we are all very busy.”
Cho-Cho, arriving about this time, was told to leave his bag in the
house and hurry back to help gather roses.
The curious neighbor departed, and Cho-Cho and the Fairy
worked for an hour, gathering flowers and vines, to make the
beautiful house more beautiful.
At last it was all finished, and the Fairy hurried upstairs to don her
party gown before the guests should arrive.
When she came down the stairs a half hour later, Cho-Cho, waiting
in the hallway, held his breath in admiration, for the little Fairy was
so lovely that words can scarce describe her. Her gown fell in graceful
lines about her slender figure—neck and arms were bare, and her
flower-like face crowned with its shining hair was radiant with joy.
Forget-me-nots were twined among her curls, and she carried a
bunch of them in her shapely hands. No jewels adorned the Fairy,
but in every way she expressed simplicity and grace.
Cho-Cho, coming forward, dropped lightly on one knee, and taking
the Fairy’s hand, raised it to his lips.
“You are wonderful,” he said.
 “You like my gown, Cho-Cho?” she questioned, and smiled upon
him, for these two were old and tried friends.
A noise from without told of the advent of a guest, and Health
went forward to greet the visitor.
It proved to be the Persian Cat, looking quite handsome in a soft-
gray hat, which he gracefully doffed to the Fairy, and he carried a
gold-headed cane.
One could well imagine him a great favorite with the ladies, for he
was indeed a finished dandy.
“You rival your flowers in beauty, Fairy,” he said, as he took her
hand.
At the noise of the garden gate closing, they looked up to behold
the Wonderful Dog and his wife coming toward them.
The Wonderful Dog had on a smart new collar, and came forward
with dignity to present his wife to the Fairy, for they had never met.
Mrs. Dog was a fluffy little thing, who seemed really to care more
about her new dress and the latest style than any other subject.
The next arrival was the little Boy from the Farm. He came timidly
toward the group, looking rather frightened, but when he spied his
old friend, Cho-Cho, all his fears vanished and he smiled with
pleasure.
They were busily chatting in the Rose Arbor, when the little
Vegetable Men came softly in and stood beside the Fairy.
She welcomed them with kindness, for these were modest little
people and felt rather strange among the handsome company.
With a whirr of wings and a flash of color, the Lovely Bird and his
brother Rumor were among them and congratulated the Fairy on the
perfect weather she was having for her Fête.
The company were all assembled now except the Red Brown
Squirrel, and the Fairy asked if anyone had seen him on the high-
road as they came hither. But no one had heard of him, although the
Persian Cat had come from that direction.
An hour slipped by. The Fairy was growing anxious and Cho-Cho
repeatedly looked at his watch.
At last he said:
 “With your permission, Fairy, I will go look for the Red Brown
Squirrel. He was my friend and I greatly fear some evil has befallen
him.”
“I, too, will go,” said the Cat, “there are many dangers upon the
high-road, and a comrade may be needed.”
“I am with you,” said the Dog. “Lead on, Cho-Cho.”
“Friends,” said the Persian Cat, “I believe I can find the Red Brown
Squirrel. Between his home and the Fairy’s garden there lives an evil
imp, called Jealousy. I passed him today as I journeyed hither and he
scowled upon me with a look of rage. He was afraid to attack me, but
the Squirrel is small and not a match for this cruel imp, and has
doubtless fallen a victim to his malice.”
“Hasten, hasten,” said Cho-Cho, and passing through the gate he
started briskly toward the Squirrel’s home.
The Cat and the Dog followed and all three were soon lost to the
view of the anxious company in the Fairy’s garden.
The friends covered the ground rapidly and were nearing the home
of the Red Brown Squirrel, when Cho-Cho’s sharp eyes spied a half-
eaten nut lying in the roadway.
There were no trees near, and they knew that it must have been
dropped by their friend.
Looking eagerly upon the ground, they discovered the tiny
footprints of the Squirrel and the larger ones of the flat-footed imp,
leading into a field of tall grass.
Here the Cat went forward, for his sharp eyes could distinguish the
footprints with great ease. Following these signs they crossed the
field and came out upon an unused road that sloped downward, until
they found themselves beside a deep river which seemed impossible
to pass.
“Get upon my back,” said the Wonderful Dog, “the River is my
comrade, it will not harm you.”
With ease and confidence he swam forward, and carried them to
safety on the further bank.
Cho-Cho and the Persian Cat stepped from the Dog’s back and
turned to thank him, when from behind a large rock, the ugly imp
darted out and sprang toward Cho-Cho, trying to push him into the
river. But the Cat, catching the imp by the neck, held him firmly, and
he was powerless to do aught but scream.
Binding him fast they went behind the great rock and found the
Red Brown Squirrel in chains.
The Cat set him free and the friendly Squirrel stood up before
them brave and unhurt.
Then they hurried back to the Fairy and were received with relief
and pleasure.
And now, the Fairy with the Wonderful Dog leads the way to the
dining-room. The Persian Cat gracefully offers his arm to little Mrs.
Dog. Cho-Cho and the Boy come hand in hand, followed by the
Lovely Bird and Mr. Beet. The Red Brown Squirrel is telling the story
of his adventure to Mr. Onion, and Rumor and Mr. Carrot bring up
the rear.
The dining-room is a mass of roses and smilax, and in the center, a
table full of all the delicious things that children like.
Mrs. Dog remarks that the decorations are lovely, and the Persian
Cat replies that the ladies are more beautiful than the flowers.
The little Fairy flits here and there among her guests, seeing, with
charming courtesy, that all their wants are well supplied, and when
at last they can eat no more, Cho-Cho rises, and with a glass of
sparkling lemonade proposes a toast:
“To our charming hostess, the Fairy Health.”
With a right good will the company drink the toast, then led by the
Persian Cat they leave the house and with laughter and jest pass by
lovely garden paths out to the fountain—and so the fun goes on,
ending at last with a dance in the dell, and joy and happiness in every
heart.
 The Macmillan Company has arranged to publish a series of
health books for the CHILD HEALTH ORGANIZATION of
America

Health in
Education
Education in
Health

The Child Health Organization has undertaken a nation-

wide campaign to raise the health standard of the school child.
The following books are now ready:
Rosy Cheeks and Strong Heart
Rhymes of Cho-Cho’s Grandma
Cho-Cho and the Health Fairy
Child Health Alphabet

Price list furnished upon application

Order from any of the following addresses of
THE MACMILLAN COMPANY
New York
64 Fifth
Avenue

Chicago
25th St. &
Prairie Ave.
 San Francisco
609 Mission
Street

Boston
Huntington
Chambers

Dallas
330 S. Harwood
Street
Literature published separately by the CHILD HEALTH
ORGANIZATION of America
Weight Card—Tags—Posters
Health in Play—My Health Book
Standards of Nutrition and Growth
The Demonstration and Its Application
The Nutrition Class
Alphabet Cards. A to Z
Health Plays for School Children
Four Plays Dramatizing “Cho-Cho and Health Fairy”
Stories
Happy’s Calendar
Milk, the Master Carpenter
Cho-Cho’s Health Game

Price list furnished upon application

Order from the CHILD HEALTH ORGANIZATION of America
Penn Terminal Building, 370 Seventh Avenue, New York City

THE U. S. BUREAU OF EDUCATION

has arranged for the sale of the following Reprints:
Class Room Weight Record
Right Height and Weight for Boys
Right Height and Weight for Girls
Wanted! Teachers to Enlist for Health Service
Diet for the School Child
 Summer Health and Play School
Teaching Health
Child Health Program for Parent-Teacher Associations and
Women’s Clubs
Further Steps in Teaching Health
The Lunch Hour at School
Suggestions for a Program for Health Teaching in the
Elementary Schools
Your Opportunity in the Schools
Health Training for Teachers
Orders for these should be sent to: The Superintendent of
Documents, Government Printing
Office, Washington, D. C. (Remittance must accompany all
orders.)

CHILD HEALTH ORGANIZATION of America

Board of Trustees
Dr. L. Emmett Holt, President
Dr. Thomas D. Wood, Vice-President
Dr. Frederick Peterson, Secretary
James G. Berrien, Treasurer
Mrs. John Collier
Clinton H. Crane
Dr. Samuel McC. Hamill
Dr. Royal S. Haynes
Dr. Victor G. Heiser
Owen R. Lovejoy
Mrs. Frederick Peterson
Dr. Bernard Sachs
Mrs. Frank A. Vanderlip
Dr. Philip Van Ingen
Allan Wardwell
Miss Florence Wardwell
Dr. Herbert B. Wilcox
Dr. C.-E. A. Winslow
 STAFF

Sally Lucas Jean, Director

Associate Directors: Marie L. Rose Anne L. Whitney Anne

Raymond, Field Representative
Assistants: Grace T. Hallock Margaret C. Carey Alice F.
Loomis
Advisory Directors: J. Mace Andress Lucy Oppen Mabel
Bragg
 TRANSCRIBER’S NOTES
Typos fixed; non-standard spelling and dialect
retained.
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