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Chapter 1. Impacts of Agricultural Practices on Subsurface Microbial Ecology

Chapter 1. Impacts of Agricultural Practices on Subsurface Microbial Ecology

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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).



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).



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-



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







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-





Recharge area






A horizon

B horizon

unsaturated zone

capillary water

Flow path taken by



topography determined by


adjacent eievational

saturated zone

water in unconnected pores

water in chemical combination

with minerals


soil, vegetation

and both surface

and subsurface





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


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



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



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



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"


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


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


Agertved er uI. ( 1992)

Albrechtson and

Winding (1992)

Amy ef a/. (1992)


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


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


Sand and gravel acquifer sediments and

water from I to 2-m depth

Also, TCE-cometabolizing phenol


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


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


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.


Fredrickson er al.

( I991 b)

Haldeman et a/. ( 1993)

Hazen et ui. ( I99 I )

Holm et al. ( 1992)

Hopkins et al. ( I 993a)



Table I-Continued

Microbiological group

Also. TCE-cometabolizing phenol


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


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


Hopkins et al. (1993b)

Kieft el a/.( 1993)

Konopka ( 1993)

Konopka and Turco


Lindqvist and Bengtsson


Madsen et a/.( 1991 )

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