Advances in agronomy volume 54

DVANCES IN

Lgronomy

V O L U M5 E4


Advisory Board
Martin Alexander

Eugene J. Kamprath

Cornell University

North Carolina State University

Kenneth J. Frey

Larry P. Wilding

Iowa State University

Texas A&M University

Prepared in cooperation with the
American Society of Agronomy Monographs Committee
P. S. Baenziger
J. Bartels
J. N. Bigham
L. P. Bush

M. A. Tabatabai, Chairnuan
R. N. Carrow
W. T. Frankenberger, J .
D. M. Kral
S. E. Lingle

G. A. Peterson
D. E. Rolston

D. E. Stott
J. W. Stuck


Edited by

Donald L. Sparks
Department of Plant and Soil Sciences
University of Delaware
Newark, Delaware

ACADEMIC PRESS, INC.
Harcourt Brace & Company
San Diego New York Boston

London Sydney Tokyo Toronto


This book is printed on acid-free paper.

@

Copyright 0 1995 by ACADEMIC PRESS, INC.
All Rights Reserved.
No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopy, recording, or any information
storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc.
A Division of Harcourt Brace & Company
525 B Street, Suite 1900, San Diego, California 92101-4495
United Kingdom Edition published by
Academic Press Limited
24-28 Oval Road. London NWl 7DX
International Standard Serial Number: 0065-2 1 13
International Standard Book Number: 0- 12-000754- 1
PRINTED IN THE UNITED STATES OF AMERICA
95 96 9 7 9 8 99 O O Q W 9 8 7 6

5

4

3 2 1


Contents
CONTRIBUTORS
......................................................
PREFACE

..............................................................

ix

xi

IMPACTS OF AGRICULTURAL
PRACTICES
ON SUBSURFACE MICROBIAL ECOLOGY

Eugene L . Madsen
I. Introduction and Scope .........................................
I1. Subsurface Microbial Ecology ...................................
111. Agricultural Practices and Their Impact on Subsurface Habitats ....
Iv. Impact of Agricultural Practices on Subsurface Microbial Ecology . .
V. Concluding Remarks ............................................
References .....................................................

HERBICIDE-RESISTANT

FIELD

CROPS

Jack Dekker and Stephen 0. Duke
...................................................

I . Introduction
I1. Mechanisms of Herbicide Resistance

i

5
35
46
56
57

.............................

111. Selection for Herbicide-Resistant Variants ........................
rv. Herbicide-Resistant Crops by the Herbicide Chemical Family .....
V. Summary ......................................................
References .....................................................

69
71

77
80
100
101

ACIDSOIL TOLERANCE
IN WHEAT

Brett F. Carver and James D . Ownby
I . T h e Problem: Causes. Symptomatology. and Severity .............
I1. Physiology of Aluminum and Manganese ‘Tolerance in Wheat .....

I11. Genetic Mechanisms of Tolerance to Acid Soils ...................
Iv. Breeding for Acid Soil Tolerance ................................
v. Sustainable Production in Acid Soils .............................
VI. Conclusions ....................................................
References .....................................................

V

117
124
136
146
161
162
164


vi

CONTENTS

MICROBIAL
REDUCTIONOF IRON.
MANGANESE. AND OTHER METALS

Derek R . Lovley
I . Introduction ...................................................
Fe(II1) and Mn(rV) Reduction ...................................
Uranium Reduction .............................................
Selenium Reduction ............................................

Chromate Reduction ............................................
VI . Microbial Reduction of Other Metals ............................
VII . Conclusions ....................................................
References .....................................................
11.
111.
IV.
V.

176
176
202
205
210
216
216
217

NITRIFICATION
INHIBITORSFOR AGRICULTURE.
HEALTH.
AND THE ENVIRONMENT
I.
I1.
111.
I v.
V.
VI .
VII .

Rajendra Prasad and J . F. Power
Introduction ...................................................
Nitrification Inhibitors ..........................................
N l s . NI I;/NO; Ratios. and Plant Growth .......................
NIs and Crop Yields ............................................
Phytotoxicity of NIs ............................................
Health and Nitrates .............................................
NIs and Environnient ...........................................
References .....................................................
PRODUCTION AND

234
235
243
246
252
254
262
269

BREEDINGOF LENTIL

F.J . Muehlbauer. W.J. Kaiser. S . L . Clement. and R .J . Summerfield
284
1. Introduction ...................................................
285
I1. Background ....................................................
286
I11. Origin. Taxonomy. Cytology. and Plant Description ..............
291

IV. Production of Lentil ............................................
V. Fertilization and Weed Control ..................................
296
297
VI . Principal Uses ..................................................
298
VII. Major Constraints to Production ................................
.........................................
303
Hybridization
Methods
VIII .
..............................................
307
Genetic
Resources
IX .
Genetics
.......................................................
308
X.
317
XI . Interspecific Hybridization ......................................
VIII . Methods Used for Lentil Breeding ...............................
318


CONTENTS

ix.

Breeding Objectives ............................................
X. Summary ......................................................
References .....................................................

vii
321
326
327

USE OF APOMIXIS IN CULTIVAR DEVELOPMENT
I.
I1.
I11.

rv.
v.

VI.

Wayne W. Hanna
Introduction ...................................................
T h e Gene(s) Controlling Apomixis ..............................
Breeding .......................................................
impact on Seed Industry ........................................
International Impact ............................................
Evaluation .....................................................
References

.....................................................

INDEX ............................................................

333
334
337
345
346
347
347
351


This Page Intentionally Left Blank


Contributors
Numbers in parentheses indicate the pagcs on which the authors’ contributions begin.

BRETT F. CARVER (I 17), Department OfAgronomy, Oklahoma State University, Stillwater, Oklahoma 74078
S. L. CLEMENT ( 2 8 3 ) , United States Department of Agriculture, Agriailtural
Research Service, Regional Plant Introduction Station, WashingtonState University, Pidlman, Washington 991 64
JACK DEKKER (69), Agronomy Department, Iowa State University, Ames,
Iowa 5001I
STEPHEN 0. DUKE (69), United States Department of Agrinclture, Agricultural Research Service, Southern Weed Science Laboratory, Stoneville, Mississippi 38776
WAYNE W. HANNA ( 3 3 3 ) , United States Department of Agriculture, Ap’ailtziral Research Service, Coastal Plain Experiment Station, Tifon, Georgia 31 793
W. J. KAISER ( 2 8 3 ) , United States Department of Agriculture, Agricultural Research Service, Regional Plant Introduction Station, Washington State University,
Pullman, Washington 991 64
DEREK R. LOVLEY (17 S), Water Resozcrces Division, United States Geological
Survey, Reston, Virginia 22092

EUGENE L. MADSEN (l), Division of Biological Sciences, Section of Mimobiology, Cornell University,Ithaca, New York 14853
F. J. MUEHLBAUER ( 2 8 3 ) , United States Department of Agriailtzire, Agricultural Research Service, Grain Legiime Genetics and Physiology Research Unit,
Washington State University, Piillman, Washington 991 64
JAMES D. OWNBY (1 17), Department of Botany, Oklahoma State University,
Stillwater, Oklahoma 74078
J. F. POWER ( 2 3 3), United States Department of Agricziltiire, Agricultural Research Service, Universityof Nebraska, Lincoln, Nebraska 68583
RAJENDRA PRASAD ( 2 3 3 ) , Division of Agronomy, Indian Agrikziltural Research Institute, New Dehli, India
R. J. SUMMERFIELD, ( 2 8 3 ) Department of Agriculture, Plant Environment
Laboratoi-y, University of Reading, Berkshire RG2 9AD, United Kingdom

ix


This Page Intentionally Left Blank


Preface
This book is the 54th volume of Advances in Agronomy. Under the excellent editorships of A. G. Norman and N. C. Brady, this venerable serial publication has
included state-of-the-art and classic reviews over the years. The excellent quality
of Advances in Agronomy and its recognition by scientists as a first-rate reference
source continues. I am pleased to report that in a recent Science Citation Index
Journal Citation Report, Advunces in Agronomy was ranked Number 1 in Agriculture. In addition, we are publishing at least two volumes per year, which means
that reviews are published on a timely basis.
Volume 54 contains seven excellent reviews that cover some important and
contemporary topics in the crop and soil sciences. Chapter 1 is a comprehensive
and timely review on the impacts of agricultural practices on subsurface microbial
ecology. Chapter 2 also addresses a topic of much interest in the area of the environment, herbicide-resistant crops. Chapter 3 provides a thorough review on acid
tolerance of wheat. Chapter 4 addresses microbial reduction of iron, manganese,
and other metals, a topic that is of much interest to scientists. Chapter 5 covers
nitrification inhibitors with particular emphasis on their impacts on health and the

environment. Chapter 6 is a review of production and breeding of lentil, an important crop in many parts of the world. Chapter 7 addresses an important subject
in plant improvement and production, use of apomixis in cultivar development.
I appreciate the excellent contributions from the authors.
DONALD
L. SPARKS

xi


This Page Intentionally Left Blank


m A C T S OF AGRICULTURAL
PRACTICES
ON SUBSURFACE
MCROBIAL
ECOLOGY
Eugene L. Madsen
Division of Biological Sciences
Section of Microbiology
Cornell University
Ithaca, New York 14853

I. Introduction and Scope
Background Definitions
11. Subsurface Microbial Ecology
A. Structure of the Habitat
B. Function
111. Agricultural Practices and Their Impact on Subsurface Habitats
Types of Agricultural Practices

IV.Impact of Agricultural Practices on Subsurface Microbial Ecology
A. A Historical Perspective for Inquiry into Subsurface versus Surface
Habitats
B. How Can the Impacts of Agricultural Practices on Subsurface Microbial
Ecology Be Measured?
C. Measures of the Impact of Agricultural Practices on Subsurface
Microorganisms
V. Concluding Remarks
References

I. INTRODUCTION AND SCOPE
Impetus for writing this chapter arises from the convergence of two major concerns of society. The first is an awareness, developing most prominently during
the 1980s. that groundwater systems, including subsurface aquifer sediments and
vadose zone formations, are precious yet vulnerable resources worthy of both protection and scientific investigation. The second is a realization that agricultural
practices may carry with them unforeseen negative consequences such as erosion
(Morgan, 1979; Rose, 1985), biomagnification of pesticides (Carson, 1962; Moriarty, 1977), and nitrate pollution of groundwater (Spalding and Exner, 1993).
1
Advonces in Agmnoriq. Vor’ume 14
Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.


2

E. L. MADSEN

This chapter’s purpose is to reveal what is and what is not presently understood
about how agricultural practices influence microorganisms in the groundwater
habitats. Of primary importance to this purpose is recognition that physical manipulations of the Earth’s surface that are practiced by humans in order to produce
food (i.e., agricultural practices) are likely to influence subsurface microbial communities as they carry on their normal ecosystem processes beneath the Earth’s
surface (i.e., subsurface microbial ecology). Furthermore, the relationship between agricultural practices and subsurface microbial ecology is one of the “impacts.” Implicit in this latter term is the fact that microorganisms are responsive

to changes in their surroundings and that the responses may take many forms.
As is evident from the Table of Contents for this chapter and Fig. 1, both subsurface microbial ecology (depicted as the land surface in Fig. 1) and agricultural
practices (depicted as a bounding sphere in Fig. 1) have their own independent
characteristics. Furthermore, the domains of subsurface microbial ecology and
agricultural practices are discontinuous in time and space. Nonetheless, as will
become evident in the course of this chapter, points of contact between agricultural practices and subsurface microbial ecology (marked by cross-hatched craters
in Fig. 1) have the potential to be very significant. To date, however, very little
scientific research has directly addressed this important interdisciplinary subject.
This chapter develops the scheme shown in Fig. 1 by defining subsurface microbial ecology (emphasizing its unperturbed status and responsive capabilities), then
by defining agricultural practices (emphasizing mechanisms for influencing the
subsurface habitats beneath), and finally by addressing documented or yet-to-be
documented interactions between agricultural practices and subsurface microbial
ecology. In order to achieve these goals, several pertinent review articles and/or
books are frequently referenced both implicitly and explicitly. In the area of subsurface microbiology, these include Chapelle (1993); Madsen and Ghiorse ( 1 993);
Matthess et al. (1992); and Pederson ( 1 993). In the area of agricultural ecology,

Figure 1. Dynamic bounding sphere metaphor for the impacts of agricultural practices on subsurface microbial ecology (see text for explanation).


SUBSURFACE MICROBIAL ECOLOGY

3

these include Briggs and Courtney (1985); Carroll et a/. (1990); Soule er al.
(1990); and Tivy (1990). In the area of hydrogeology, these include Davis and
Dewiest (1966); Domenico and Schwartz (1990); Freeze and Cherry (1979); and
Nachtnebel and Kovar (1991).

BACKGROUND
DEFINITIONS

The terrestrial subsurface habitat and its synonym, the groundwater habitat,
reside directly beneath all continental portions of the globe. To access the terrestrial subsurface, one must excavate or drill through surface materials comprised
of soil or rock (in upland areas) or freshwater and sediments (in aquatic areas). It
is this vertical stratification of the Earth’s surface and its implicit gradation of
exposure to climatic and biological influences that makes surface and subsurface
habitats distinctive. In descending from the surface of the earth through soil, one
typically encounters materials in the following vertical sequence: the A and B soil
horizons; the C soil horizon, from which the other soil horizons may have been
derived (Brady, 1990); an unsaturated (or vadose) zone (that begins with the C
soil horizon and ends at the water table); and a capillary fringe zone residing
directly above a saturated zone which may extend through many different geologic strata (Fig. 2; Madsen and Ghiorse, 1993). Where does the surface habitat
end and the subsurface begin? For the purposes of this chapter, the groundwater
habitat begins immediately below the B soil horizon where soil scientists traditionally have felt that major biological activity ceases (Alexander, 1977; Brady,
1990; Madsen and Ghiorse, 1993). However, the transition between soil and
groundwater habitats is not delineated by soil horizons per se because the demarcation is gradual. But regardless of the type of overlying material (be it soil, rock,
or freshwater bodies and sediment), the subsurface occurs where the influences of
climate, animals, and plant roots diminish and these are replaced by predominantly hydrological, geochemical, and microbiological influences. Although freshwater habitats represent a relatively small proportion of the continental surface
area [<2% of global surface area (Wetzel, 1983) vs 24% of global surface area
for forested or cultivated soil (Ehrlich er al., 1977)], free passage of water from
the subsurface to lakes and stream and vice versa may have significant hydrological and biological impact. This may be especially true for agricultural lands because these components of the terrestrial habitat are frequently adjacent to fresh
waters (Blum e f al., 1993; Fernandezalvarez et al., 1991; Gilbert et a/., 1990;
Tremolieres et a/., 1993).
In a hydrogeologic sense, groundwater refers to water that is easily extractable
from saturated, highly permeable geologic strata known as aquifers (Davis and
Dewiest, 1966; Domenico and Schwartz, 1990; Freeze and Cherry, 1979). Be-


4

E. L. MADSEN

cause the water in these high yielding formations are major sources of drinking
and irrigation water, aquifers are of principal concern in water management and
conservation programs. Most groundwater is interstitial-it resides within a matrix of sediments and/or minerals with variable porosity, chemistry, and degree of
saturation. Although water may be readily obtained only from aquifers, the vertical profile in a typical landscape reveals a continuum of water tensions and availabilities. Water may exist in many forms: as part of the crystal matrix of minerals
in rocks; in unconnected pores of solid rock; in connected pores in solid rock; in
the saturated portions of aquifers; and in unsaturated strata as liquid gaseous and
solid phases (the latter only under extremely cold climatic conditions) in capillaries and pores. To microorganisms which, by definition, live in microhabitats, all
available forms of water, except those in chemical combination with minerals,
may be important. Therefore, this chapter uses a broad definition of groundwater
which includes capillary water, water vapor, and water within aquifers (Madsen
and Ghiorse, 1993). As a habitat for microorganisms, the subsurface includes the
unsaturated zone because it may contain significant amounts of biologically available water. Also, unsaturated zones may be transiently saturated during recharge
events and they may influence both the chemistry and microbiology of the saturated zone. In summary, throughout this chapter, groundwater refers to all subsurface water found beneath the soil A and B horizons that is available to sustain and
influence microbial life in the terrestrial subsurface (Ghiorse and Wilson, 1988;
Madsen and Ghiorse, 1993).
Agriculture is derived from Latin “agri cu1tura”-meaning “cultivation of the
land.” Agriculture is the science, art, and business of cultivating the soil, producing crops, and raising livestock useful to humankind. As stated by Tivy (1990),
the principal resource base for agriculture is the physical environment. The primary process in agriculture is photosynthesis-a process that has not yet been
replicated outside living chloroplast-containing cells of green plants. Thus, the
cultivated crop plant is the basic “production unit” of agriculture because of its
ability to manufacture complex organic compounds from inorganic materials supplied by the atmosphere and soil. The challenge of agriculture is to efficiently
manage the physical environment to provide for the biological demands of the
crop plant. Tivy (1990) insightfully has written that this linkage between crop and
environment is established through the management practices which: (1) select the
desired crop to match the prevailing climatic and edaphic conditions; ( 2 ) propagate the crop via tillage practices that favor proper crop germination and growth;
and (3) protect the crop from competition with weeds for growth-related resources
(light, COz, water, nutrients) and from yield reduction by animal pests and plant
pathogens.

SUBSURFACEMICROBIAL ECOLOGY

5

11. SUBSURFACE MICROBIAL ECOLOGY
OF THE HABITAT
A. STRUCTURE

1. Hydrology and Geology

The terrestrial subsurface is an important component of the landscape through
which water passes as it cycles among the atmosphere, soil, lakes, streams, and
oceans (Fig. 2, Section LA). Once water has infiltrated below the surface layer of
soil, it has several possible fates. It may (i) return to soil via capillary, gaseous, or
saturated transport; (ii) be intercepted by plant roots; (iii) reach streams, lakes, or
ponds via saturated flow; (iv) reverse its saturated flow direction from streams
or lakes back into subsurface strata when levels of surface waters are high;
(v) directly reach the ocean via saturated flow; (vi) become mixed with seawater
when groundwater withdrawal in coastal areas causes seawater to intrude inland;
or (vii) enter a closed deep continental basin (Fig. 2 ; Domenico and Schwartz,
1990). Regardless of the flow path taken through the subsurface, groundwater
remains in the biosphere. However, the residence time before water exits the subsurface is highly variable. Return of subsurface water to the soil may occur within
a few days or weeks, though return from a deep continental basin may require
thousands of years (Madsen and Ghiorse, 1993; Freeze and Cherry, 1979).
In conceptualizing the routes taken by water through the terrestrial segment of
the hydrologic cycle, Chapelle’s presentation (Chapelle, 1993) of local, intermediate, and regional flow systems is insightful. Chapelle provides the following
definitions for these three flow systems based on relationships among surface topography, large-scale geological structures, and the depth of water penetration
along its path from recharge to discharge areas: ( 1 ) A local system has its recharge
area at topographic high and its discharge area at a topographic low that are located adjacent to each other; (2) an intermediate system occurs when recharge and

discharge areas are separated by one or more topographic highs; and (3) in a regional system, the recharge area occupies the regional water divide and the discharge area occurs at the bottom of the basin. Figure 2, which incorporates
Chapelle’s flow systems (Chapelle, 1993), illustrates the spatial and functional
relationships between the geological setting of the subsurface and its most dynamic component, water.
Beneath the soil which, by definition, is the zone of pedogenesis, lie the unsaturated and saturated subsurface zones. This view of the subsurface habitat as being
delineated in terms of the degree to which water occupies voids in a porous matrix
(if air has been completely displaced by water, the system is “saturated”; if not,
the system is “unsaturated”) is satisfying, but it is also simplistic. For superim-


6

E. L. MADSEN

Preclpitatlon
Evaporation

Recharge area

Infiltration

Stream

Ocean

-

Soil
A horizon
B horizon

unsaturated zone
capillary water

Flow path taken by
groundwater

V

topography determined by

-

adjacent eievational

saturated zone

water in unconnected pores

water in chemical combination
with minerals

Destination:
soil, vegetation
and both surface
and subsurface
water
bodies

extremes

m:
recharge and discharge

areas are separated by one
or more elevational maxima

Path 01 water Is deep
beneath other flow systems;
flow path connects highest
elevation of regional
recharge area to lowest
discharge point 01 regional
basin.

Figure 2. Conceptual Row system for understanding the role of the soil and the subsurface habitat
in the hydrologic cycle. (Figure from Madsen and Ghiorse (1993) modified according to flow system
categories by Chapelle (1993) and groundwater categories by Domenico and Schwartz (1990)l.

posed upon the degree of water saturation are the geological, geographic, and
climatic characteristics. At a given location on the Earth’s surface, the stratigraphy
beneath reflects a unique and complex history of geological, hydrological, and
chemical events (e.g., sedimentation, erosion, volcanism, tectonic activity, dissolution, precipitation, and biogeochemical activity). The result often is a heterogeneous geologic profile whose complexity may be compounded by variations in
pore water chemistry that may stem from localized aberrations in mineral phases


SUBSURFACE MICROBIAL ECOLOGY

7

or inorganic or organic solute concentrations. The large surface area provided by

rocks and sediments in the porous matrix may strongly influence the physical and
chemical conditions of the groundwater habitat by altering concentrations of dissolved aqueous constituents at the surfaces and by sorbing microbial cells (Madsen and Ghiorse, 1993; van Loosdrecht et al., 1990). Sorption and aqueous equilibrium reactions are most likely to be influential in the saturated zone. But many
subsurface habitats are dominated by unsaturated zones as well. In arid climates,
the unsaturated zone may be hundreds of meters deep. Rainfall in such desert
climates may be insufficient to allow saturated infiltration of soil to reach to the
water table, except in restricted low-lying areas (Davis and Dewiest, 1966).
Therefore, rather large area portions of deserts may have unsaturated zones beneath them with little or no saturated water flux. Under such circumstances, vapor
phase reactions may be the prevalent form of geochemical change. Such conditions have important implications for agricultural irrigation practices as well as
both microbial physiology and activity (see Sections II.A.2, II.B, 111, and 1V.C).
Freeze and Cherry (1979) have presented the idea of “chemical evolution” of
groundwater as it passes from the atmosphere in recharge zones along the variety
of flow paths such as those depicted in Fig. 2 and described in Section 1I.A. I. As
precipitation, water begins as pure distillate containing only atmospheric gaseous
and atmospheric particulate materials. After contact with soil and deeper subsurface sediments, the chemical composition of the water changes Substantially. Not
only do components in surface and subsurface matrixes dissolve, volatilize, and
precipitate, but, as the water reaches zones that are more remote from the atmosphere, complexation and oxidation/reduction reactions also occur. Many of the
reactions are strictly geochemical (Chapelle, 1993; Domenico and Schwartz, 1990;
Stumm and Morgan, 1981; Morel and Hering, 1993; Schwarzenbach et al., 19931,
but many are also microbiologically mediated (see Sections 1I.B and 1V.C).
The chemical composition of a given sample of groundwater reflects the integrated history of chemical and biochemical reactions that occur along a given flow
path through soil and geologic strata. Because of the diversity of flow paths and
biogeochemical reactions, the composition of groundwater is quite variable. Nonetheless, some generalizations can be made. In aquifers used for drinking water
supplies that are not influenced significantly by human activity, major chemical
constituents (>5 mg/liter) typically include calcium, magnesium, silica, sodium,
bicarbonate, chloride, and sulfate while minor constituents (0.01- 10 mgniter) include iron, potassium, boron, fluoride, and nitrate; with trace amounts (<0.1 mg/
liter) of many inorganics and organics (including humic acids, fulvic acids, carbohydrates, amino acids, tannins, lignins, hydrocarbons, acetate, and propionate
(Domenico and Schwartz, 1990). However, as discussed in Sections III and IV,
human activities (including septic systems, landfills, other types of waste disposal,
and agricultural practices) may alter the chemistry of groundwater substantially
by adding high concentrations of solutes such as both toxic and nontoxic organic

8

E. L. MADSEN

carbon compounds and nutrients. Detailed descriptions of geological and geochemical principles and the hydrogeologic properties of the Earth’s crust are beyond the scope of this chapter. For these, readers are referred to Domenico and
Schwartz (1990), Freeze and Cherry (1979). Larson and Birkeland (1982), Morel
and Hering (1993), Stumm and Morgan (1981), and Strahler (1984).

2. Organisms
The subsurface biological community is unique in that it consists primarily of
unicellular bacteria, fungi, and protozoa. Though algae may be present under
some circumstances (Madsen and Ghiorse, 1993), absence of sunlight severely
limits the activity and significance of any photosynthetic microorganisms that may
be transported into aquifers from adjacent habitats. Larger organisms (such as
fish) occur only rarely in subterranean caves or cavernous aquifers that are connected to the surface by suitably large channels or fissures (Ghiorse and Wilson,
1988; Hynes, 1983; Longley, 1981). Other aquatic fauna, such as amphipods, may
inhabit ecotone habitats such as riverbank or lake sediments that are transitional
to the subsurface (Danielpool er al., 1991; Gilbert et al., 1990).
The accumulating evidence indicates that most of the known physiological
types of bacteria that we have come to expect in moderate (as opposed to extreme)
marine and freshwater habitats on the surface of Earth are also present in moderate
subsurface habitats. Ghiorse and Wilson (1988) and later Madsen and Ghiorse
( 1993) compiled extensive lists of reports examining microorganisms and their
potential metabolic activities in subsurface habitats. A variety of experimental
techniques performed on samples from many sites have revealed wide-ranging
metabolic capabilities of both aerobic and anaerabic microorganisms. KolbelBoelke et al. (1988) listed physiological properties of 2700 aerobic heterotrophic
bacteria isolated from a Pleistocene sand aquifer in Northern Germany. Also,
Balkwill ( 1 990) tabulated 27 metabolic groups or physiological types of aerobic

and anaerobic bacteria isolated from a single deep aquifer drilling site in South
Carolina. Similarly,Haldeman et al. ( 1 993) isolated and characterized 2 10 aerobic
bacteria from freshly exposed rock faces in a vadose zone tunnel system 400 m
beneath the Nevada test site. Additionally, Kampfer er al. (1993) isolated and
characterized 3446 aerobic and anaerobic bacteria from a shallow contaminated
aquifer where a large-scale bioremediation project was underway. To further illustrate the breadth of metabolic diversity found among subsurface microorganisms
in shallow and deep aquifer systems, results of recent studies (published since
1991) in which the physiological types or activities of subsurface microorganisms
were determined have been summarized (Table I). In evaluating the meaning of
these results, it is important to recognize the distinction between metabolic potential, determined by incubating field samples in the laboratory, and in siru microbial
activity. This distinction between what can be measured in laboratory-incubated


SUBSURFACEMICROBIAL ECOLOGY

9

experimental samples and the actual expression of metabolic activity in field sites
has been extensively discussed by Madsen et al. (1991), Madsen (1991), Madsen
and Ghiorse ( 1993), the National Research Council ( 1993), and Madsen ( 1995).
Despite this caveat, it is clear from Table I that the metabolic diversity of subsurface microbial communities, like those of all other moderate habitats in the
biosphere, is substantial. This diversity has major implications for all aspects of
groundwater microbiology, especially for the responses of subsurface microorganisms to agriculture-induced change (see Sections 111 and IV).
Surface soils in temperate regions typically contain lo* to lo9 bacteria per
gram. The major chemical and physical influences which govern bacterial abundances in soil [available organic carbon, nitrogen, phosphorus, sulfur, moisture,
pH, electron acceptors, grazing by predators, immigration of microorganisms
from other habitats, etc. (Alexander, 1977)] are modified in the subsurface along
the hydrologic flow paths. The nature of the geologic stratum (mineral type, particle size distribution, texture, hydraulic conductivity, etc.) also may determine the
abundance and distribution of bacteria in a given subsurface zone.
Madsen and Ghiorse (1993) have presented a generalized scheme for the vertical distribution and potential metabolic activity of subsurface microorganisms. In

descending from the A to B soil horizons into the C horizon, a decline in nutrient
levels is accompanied by a drastic decline in bacterial abundance. Indeed, many
reports in the older soil microbiology literature suggested that few, if any bacteria,
existed in the C horizon. The C soil horizon often marks the beginning of the
unsaturated subsurface zone, which supports far fewer bacteria than the B soil
horizon. However, the numbers of bacteria usually do not continue to diminish
with depth. Instead, microbial abundance typically increases substantially at the
water table and just above it in the capillary water zone (Fig. 2 ) . It is possible that
these interface zones between the unsaturated and saturated zones may be the site
of relatively dynamic mixing of oxygen and recently recharged nutrients in shallow unconfined aquifers. As one continues deeper through the water table into the
saturated zone, the abundance and potential activities of microorganisms generally remain high relative to the unsaturated zone. In a given locale, both the moisture regime and the type of geologic strata below the water table may be highly
varied (e.g., sediments high in clay of low transmissivity, beds of crystalline or
porous rock with varying degrees of fracturing, and zones of highly transmissive
sand and gravel may be present). Highly transmissive saturated zones (high-yield
aquifers containing water that was recharged relatively recently) typically show
microbial abundances and metabolic activity potentials that are two to four orders
of magnitude greater than those of hydrologically nontransmissive zones, whose
waters and nutrients may be relatively old and depleted. Thus, depth per se does
not govern the abundance and activity of bacteria in the saturated zone; rather, the
hydrological, physical, and geochemical properties of each stratum appear to govern the population density and degree of metabolic activity of its own community.


-

Table I
Summary of Microbiological Groups Detected in Subsurface Habitats"

0

Microbiological group

Aerobic cbemoheterotrophic bacteria

Sample type

Method of detection

Field site underlain by glacio-fluvial sand

Injection of herbicides, MCPP, and atrazine in siru into groundwater; microcosms amended with herbicides
Microscopy, viable counts on agar media,
'*CO, production from radiolabeled
acetate and phenol; [ 'Hlthymidine incorporation into cells
Viable counts on agar media; characterization of 47 isolated bacteria using
measures such as metal resistance,
phospholipid fatty acid profiles, and
carbon source utilization
[ 'JC]Glucose and toluene mineralization;
14C-labeledamino acid incorporation;
MPN, and direct microscopic counts
Six bacteria were stored in media for 100
days; cellular and physiological
changes were monitored
Random selection and isolation of 63 bacteria; these were challenged to grow under various exposures to UV radiation
and hydrogen peroxide
I4CO, production from '"C-labeled chelating agents (DTPA, EDTA, NTA)
Enrichment and isolation of 25 methanotrophic bacterial strains; phospholipid
fatty acid analyses; GC analysis of culture fluids for TCE and PCE; T O z
production from [ '"C-ITCE; DNA hybridization assays

4 to 3 1-m depths of sediments

One water sample and three vadose zone
deep rock samples from the Nevada test
site

Well water from shallow aquifer at hazardous waste site

Deep rock vadose samples from the Nevada test site
Also, UV radiation and hydrogen
peroxide-resistant bacteria

Methanotrophs capable of cometabolizing
chlorinated alphatic hydrocarbons

Deep coastal plain acquifer sediments
from depths of 150-500 m

Deep coastal plain acquifer sedimenls
from 0- to 376-m depths
Trichloroethylene- and perchloroethylenecontaminated well water from the Atlantic coastal plain

Reference
Agertved er uI. ( 1992)

Albrechtson and
Winding (1992)

Amy ef a/. (1992)

Armstrong

el a/. ( 1991 )

Amy et al. ( 1993)

Bolton et al. 1993)

Bolton e t a / . 1993)
Bowman et a/. (1 993)


Also, actinomycetes

Vadose zone paleosols at depths ranging
from 53.6 to 63.7 m; also vertical cliff
faces
Carbonate sands beneath a pentachlorophenol (PCP) wood-treating facility
Deep Atlantic coastal plain acquifer

Deep Atlantic coastal plain acquifer

Fresh rock faces in a deep subsurface vadose zone tunnel system, 400 m in
depth (Nevada)

Sediments and ground waters to depth of
550 m in southeastern Atlantic coastal
plain
Sand and gravel acquifer sediments and
water from I to 2-m depth

Also, TCE-cometabolizing phenol
degraders

Shallow confined sandy acquifer 5-m
depth interval (central California)

Microscopy/respiratory activity stain; viable counts on agar media; ATP concentration; mineralization of [ “C-Iglucose; ecological diversity indices
G U M S determination of PCP and metabolites in field samples, determination
of sediment/PCP sorption coefficients
Microscopy; growth on various compounds; “CO2evolution from toluene;
naphthalene; hybridization with pWW0
and NAH7 plasmids
Agar media, 108
physiological tests applied to 198 isolated bacteria
Microscopy, agar media, isolation and
testing of 210 bacterial strains; growth
on agar media; analysis of fatty acid
methyl esters; microbial diversity
analyses; Euclidean distance cluster
analyses
Microscopy; growth on agar media;
screening of isolated bacteria for physiological capabilities
Microscopy; ATP analysis; laboratory and
field ( i n situ) incubation of sediment
and water contained within a steel cylinder and amended with ’ H z Oand 23
organic contaminants; fluids were periodically removed from vessels and analyzed to demonstrate contaminant
losses
Injection and circulation of phenol into
aerobic groundwater at a field site

stimulated metabolism of TCE

Brockman et crl. ( I 992)

Davis et crl. ( I994a)

Fredrickson el al.
(1991a)

Fredrickson er al.
( I991 b)
Haldeman et a/. ( 1993)

Hazen et ui. ( I99 I )

Holm et al. ( 1992)

Hopkins et al. ( I 993a)

(continues)


e

Table I-Continued
Microbiological group
Also. TCE-cometabolizing phenol
degraders

Sample type

Method of detection

Shallow confined sandy aquifer 5-m depth
interval (central California)

Field and laboratory metabolism trials
demonstrated that phenol stimulates
high efficiency cometabolism of chlorinated ethenes by indigenous subsurface
microorganisms
Microscopy; growth on agar media, production of “CO, from [ ‘‘C]glucose and
-acetate; water potential measurements
performed on field samples and on
growing cultures
Isolation of bacterium able to grow on
methylanilines and aniline; oxygen uptake by cells in the presence of organic
compounds, enzyme assays; GC determination of anilines in culture media
‘“COzevolution and “ C incorporation
into biomass from [ “Clglucose, -phenol, and -aniline; microscopy, agar media; 32Pincorporation into phospholipid
Isolation of two bacterial strains; examined influence of nutrient status on distribution of bacteria between solids and
solution using a continuous flow system
I4CO2production from [ I4C]p-hydroxybenzoate, -naphthalene, and phenanthrene; enumeration of microorganisms,
including protozoan predators, inside
and outside a plume of contaminated
ground water

Deep vadose zone (2- to 450-m depths) in
the intermountain zone of the western
United States

Shallow unsaturated zone sediments to
5-m depth

Shallow unsaturated zone

7- to 8-m depth saturated zone in sedimentary strata

Shallow unconfined sandy acquifer at
depths ranging from 1 to 6 m

Reference
Hopkins et al. (1993b)

Kieft el a/.( 1993)

Konopka ( 1993)

Konopka and Turco
(1991)

Lindqvist and Bengtsson
(1991)

Madsen et a/.( 1991 )