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Title: Deep Subsurface Microbiology


1
Deep Subsurface Microbiology
Savannah River Drilling Program
Module 6 Deep Subsurface Microbiology
2
Deep Subsurface Microbiology
Deep Subsurface Microbiology 
  • Deep aquifers (hundreds or thousands of metres
    below surface) have only recently been
    investigated other than by petroleum or sulfur
    companies seeking deposits or concerned with the
    impact of microorganisms on their drilling and
    mining activities.
  • The first major project to investigate the deep
    subsurface was started in 1986 at the U.S.
    Department of Energy site at Savannah River. The
    drilling, for the first time, was carried out
    with microbial sampling as a prime objective.
    Great care was taken to maintain the drill holes
    in a state suitable for microbial sampling.
  • The Savannah River plant overlies the Atlantic
    Coastal Plain and has unconsolidated sediments to
    a depth of 300 metres. The sediments are then
    underlain by crystalline metamorphic rock or
    consolidated mudstone. There are some sandy
    aquifers interspersed between the clay and silt
    formations.
  • A sample drill hole was bored first to determine
    the stratigraphy of the site. Then a sampling
    hole was drilled. The drilling fluid (sodium
    bentonite) was used to continuously flush the
    hole as it was drilled. To prevent contamination,
    the sampling container was lowered to a depth
    below the circulating drilling fluid. Autoclaved
    or steamed stainless steel core liners were used
    to collect the samples.
  • The sediments were removed from the core liners
    in a N2-flushed glove bag to preserve anaerobic
    conditions. All transfers were done within 30
    min. of sampling the drill hole.
  •  

3
Subsurface Environments
SLIMES, or subsurface lithoautotrophic microbial
ecosystems, exist in the pores between
interlocking mineral grains of many igneous
rocks. Autotrophic microbes (green) derive
nutrients and energy from inorganic chemicals in
their surroundings, and many other microbes
(red), in turn, feed on organics created by
autotrophs.
SUBSURFACE ENVIRONMENTS vary considerably in
the composition of the surrounding rock.
Deep-living microbes pervade both oceanic and
continental crust and are especially abundant in
sedimentary formations. Such microorganisms fail
to survive only where the temperature exceeds
about 110 degrees Celsius (orange areas). The
nature of the population does, however, change
from place to place. For example, a porous
sedimentary layer that acts as a conduit for
groundwater may contain both oxygen-rich (light
blue) and oxygen-poor (dark blue) zones, and the
bacteria found within its different regimes will
vary according to the chemical reactions they use
for energy (blue bar, above).
From Scientific American http//www.sciam.com/10
96issue/1096onstottbox3.html
4
Sampling
Sampling such depths is difficult especially
when sterile or aseptic conditions have to be
maintained Tracer dyes were added to check
whether anything could have penetrated into the
cores. When the drillers brought a core to the
surface, it was encapsulated and placed it in a
glove box for processing. These were filled with
nitrogen as a precaution to protect any
obligately anaerobic bacteria.
SUBSURFACE EXPLORATION (above, left) requires a
great length of rotating steel pipe to snake
downward from a drilling derrick to an
underground target. As the pipe rotates, a
diamond-studded drill bit at the bottom of the
borehole (detail, right, bottom) cuts away at the
underlying rock and surrounds a cylindrical
sample that is later extracted when the pipe is
withdrawn. Lubricating fluid with a special
tracer substance is pumped down the center of the
pipe (detail, right, top) and out through holes
in the bit (arrows). The cylindrical rock sample
remains in place as the pipe and bit rotate
because it sits within a stationary inner barrel
that is supported by a bearing. As a core of rock
fills the inner barrel, a bag of concentrated
tracer material above it breaks open and coats
the outer surface of the sample (yellow). Cores
recovered in this way are cut into short segments
from which the outer rind marked by the tracer is
removed to avoid contamination (above, right).
From Scientific American
5
Activity 1
  • A number of different experiments were performed
    with these materials.
  • Rates of incorporation of acetate into lipids
  • Radioactive thymidine incorporation into DNA
  • Aerobic mineralisation of acetate and glucose to
    carbon dioxide
  • Anaerobic mineralisation of acetate and glucose
    to carbon dioxide
  • Most Probable Number (MPN) counts of aerobic
    heterotrophs
  • Some typical Results

Microbial Activities and MPN counts of Aerobic
Heterotrophic Microbial Populations from Deep
Subsurface Boreholes
6
Activity 2
Aerobic and Anaerobic Mineralization of
14C-acetate and 14C-glucose to 14CO2 in Deep
Subsurface Sediments
  • Some general observations were
  • The numbers of culturable bacteria in the clay
    sediments were almost 3 to 5 orders of magnitude
    (1000 to 100000 times) less than in the shallow
    aquifers or the surface soils. 
  • The sandy water-bearing layers had the highest
    counts and the greatest microbial activities. 
  • Water-bearing sandy layers had higher numbers
    and activity than clay layers much nearer the
    surface depth is not necessarily the limit to
    growth and activity.

7
Other bacteria
  • In another study, coliforms, sulfate reducers and
    methanogens were enumerated.
  • Anaerobic metabolic activity was measured by
    monitoring the disappearance of lactate, formate
    and acetate and the production of methane and
    hydrogen sulfide.
  • Although anaerobic microorganisms were present
    in the deep subsurface layers in the Savannah
    River site, the sediments in the area did not
    appear to be primarily anaerobic in nature. The
    anaerobes were 100 to 100000 times less abundant
    than aerobes. The anaerobes found were presumably
    growing in anaerobic microenvironments or were
    tolerant to oxygen levels found in the sediments.
  • Most of the anaerobes were found in the
    water-saturated sandy zones where anaerobic
    degradation of acetate and benzoate and methane
    production were found in addition to the
    metabolism of lactate and formate that was found
    throughout the sediment.
  • There was no phenol degradation.
  • The numbers of coliforms dropped rapidly from
    the surface layers to the deeper layers. There
    was no evidence of coliforms in the unused
    drilling fluids, but coliforms were found in
    circulating drilling fluids. No fecal
    streptococci were found. All of these
    observations lead to the conclusion that recent
    contamination of the deep subsurface by surface
    coliforms or by sewage is unlikely, and that the
    subsurface may harbour a population of coliform
    bacteria.
  • Another part of the investigation looked at
    denitrification in the subsurface. The acetylene
    blockage method was used to  detect
    denitrification activity. All tested samples from
    all depths showed activity it was highest at the
    surface and decreased with depth. It was also
    highest in the water-bearing sandy parts of the
    subsurface and lower in the clay sediments.
    Addition of nitrate enhanced denitrification in
    samples from immediately below the water table
    down to a sample depth of 289 metres.

8
Survival
  • How do these bacteria grow or survive at depths
    up to 1.7 miles below the surface ?
  • Possibilities
  • They were incorporated into the sedimentary rock
    materials during formation many millions of years
    ago and have survived on a "starvation" diet
    since then.
  • They enter through infiltrating groundwater from
    the surface (most likely with bacteria found in
    igneous rocks such as basalt or granite because
    of the very high temperatures during formation).
  • Some bacteria are growing slowly on inorganic
    energy sources and thus providing organic carbon
    to other microorganisms in the rock matrix (SLiME
    - Subsurface Lithoautotrophic Microbial
    Ecosystems).
  •  

9
Key Points
Key Points
  • What is meant by "Deep Subsurface microbiology"?
  • How can activities of microorganisms in the deep
    subsurface be measured?
  • What are the typical results of such measures
    and what do they mean?
  • Compare this section to the next one on
    Groundwater Microbiology - are there
    similarities?

10
Groundwater Microbiology
Module 7 - Groundwater Microbiology
  • General Overview of Groundwater
  • Overview of Microbiology
  • Sampling Methods
  • Environmental Conditions in Groundwater (7a)
  • Contaminated Groundwater (7a)
  • Movement of Groundwater (7a)
  • Movement of Contaminants in Groundwater (7a)
  • Biodegradation and Kinetics (7a)
  • Groundwater Modeling (7a)

11
General Introduction
General Introduction to Groundwater Although
groundwater is third in quantity behind the
oceans and glaciers and permanent snow, it
comprises about 69 of the world's fresh water
and about 1.7 of the world's total water.
However, its replacement time is over 1400 years,
about half that of the world's oceans. 
Groundwater can exist in many different
environments, but is important when it is in
aquifers that we can access for our needs. The
particular type, chemical content (some
groundwater is extremely alkaline and/or saline)
and depth below the surface depends on many
factors including the rock materials or substrate
it is in and the infiltration of water from other
sources. Distribution of the the world's water
Most rocks near the earth's surface are in
somewhat unstable condition over long time
periods they break down into smaller and smaller
particles and form soils. Soils are redistributed
by water transport, air movement, sedimentation,
ice and gravity and eventually form new types of
rock materials. Occurring over geologic time
periods, these processes lead to the formation of
the three main types of aquifers. Aquifers are
water-bearing reservoirs capable of yielding
usable quantities of water. The three types are
alluvial, sedimentary and glacial.
Igneous/metamorphic rocks are formed by volcanic
activity or heat due to pressure and can contain
water-bearing rock materials (aquifers).
Alluvial Rivers and streams form groundwater
reservoirs consisting of alluvial deposits. The
rivers carry and deposit rock materials on the
flood plain. These deposits are often of uniform
grain size due to the action of the river
currents sorting the particle sizes upon
deposition. Some others show sharp gradations in
particle size due to differential deposition onto
a river bed at slower and faster regions of the
current flow. Aquifers can be formed in these
deposits when they are covered by other materials
and buried. Sedimentary Deposition of
sediments in marine and freshwater can lead to
sedimentary rock materials being formed. If the
land then rises due to continental movements or
volcanic activities, these rock materials can
then come to lie above current sea levels. If
porous, they can be water-bearing.
12
Hydrogeology of Canada
  • Glacial Glacial aquifers are present throughout
    much of the highly populated area of the US and
    Canada. In these cases, the underlying bedrock is
    igneous or metamorphic and has little water. If
    the bedrock does contain water in these areas, it
    is often of poor quality (brine).
  • Glacial activity "grinds" rock materials and
    deposits them at a distance from their source.
    Rock material may be deposited at the edge of the
    glacier as it retreats, causing the formation of
    moraines.
  • Some of the material in glaciers is released and
    moved as outwash as the glacier melts to water
    and forms rapidly flowing rivers.
  • There have been numerous glaciation events in
    North America leading to the complex geological
    and aquifer formations of the Great Lakes area in
    particular. 
  • Hydrogeology of Canada
  • Although surface water is abundant (about 24 of
    the surface fresh water supply of the entire
    world), about 10 of the water supplied by
    municipalities with populations of over 1000 is
    groundwater. Groundwater makes up an even greater
    proportion of the water used by individual houses
    because of the preponderance of dug and drilled
    wells in rural areas.
  • Differences in climate and geology lead to the
    six regions of different hydrogeological
    conditions in Canada. The map below (from the
    United Nations, 1976) shows these regions
  • the Cordilleran,
  • the Interior Plains,
  • the Northern,
  • the Canadian Shield,
  • the St. Lawrence and
  • the Appalachian regions.

13
Map of Canada
Groundwater in Canada
14
Regions in Canada
The Cordilleran Region is mainly crystalline
rocks with little surface deposit of materials.  
There is complex aquifer development in the river
valleys and glaciated area. The Interior
Plains Region is at the southern limit of the
permafrost between the Rockies and the Canadian
Shield region. The strata are nearly horizontal
in arrangement with a thick layer of surficial
deposits. There are some outwash type aquifers
and some bedrock types where the underlying rock
materials are water-bearing. In parts of Alberta
and Saskatchewan, the aquifers contain water with
very high salt concentrations The Northern
Region is all of Canada north of the southern
edge of the discontinuous permafrost limit.
Rainfall and snowfall is low and the area is over
a crystalline bedrock or sedimentary materials.
Permafrost occurs everywhere and the aquifers can
be on top, within or below the permafrost layer.
The Canadian Shield Region is on mixed
crystalline rocks with irregular surface
deposits. The topography is very rugged.
Groundwater aquifers are rarely used and are
limited in extent. The St. Lawrence Region is
covered with a thick surface deposit of glacial
origins. Aquifer chemistry often reflects the
limestone and dolomite rock materials i.e. they
contain calcium and magnesium bicarbonates. The
Appalachian Region is characterised by flat
sedimentary rocks, with thin surface deposits.
The higher rainfall and short flow path leads to
groundwater with lower levels of salts than the
neighbouring St. Lawrence Region.
15
Hydrologic cycle
The Groundwater Environment
Groundwater is part of the overall hydrologic
cycle Groundwater can be present close to the
surface (shallow aquifers) or at great depths.
It can be in an UNCONFINED AQUIFER where the
water is in a porous layer (e.g. glacial till in
the diagram below) or, if trapped between two
impermeable rock formations can be a CONFINED
AQUIFER. The recharge zone where water enters
the groundwater can be at a distance. These
recharge areas are now being protected where the
aquifers are used as sources of drinking water
(e.g the Region of Waterloo).
16
Confined unconfined aquifers
17
Flow
In a typical system, surface water and
groundwater interact overland flows of water
enter streams and rivers and the groundwater can
also enter or leave the rivers and streams. The
groundwater-containing zone is often called the
"saturated zone" whereas the soil above it is the
"unsaturated zone". The WATER TABLE is simply the
top of the groundwater saturated zone.
18
Artesian Well
If a confined aquifer happens to be present and
the hydraulic pressure due to its topography is
sufficient to drive the water to the surface by
simply drilling into the aquifer, it is said to
be an ARTESIAN WELL. Usually this happens because
the recharge zone is higher than the lower land
surface and the water is confined by the
impermeable rock materials so that the hydraulic
head is maintained
19
Hydraulic Conductivity
The rate at which groundwater flows through the
matrix materials is determined by the hydraulic
pressure and the conductivity of the materials.
This can be measured in gallons per day per
square foot. Typical values are given in the
diagram below for different matrix materials.
Note hydraulic conductivity is a log. scale
20
Overview of Microbiology 1
Overview of Microbiology
  • The microbiology of groundwater has only
    recently received much attention from
    microbiologists.
  • Early studies indicated a decrease in numbers
    with increasing depth, so it was assumed that
    groundwater in aquifers would essentially be
    sterile.
  • After 1970, studies began to reveal the extent
    and complexity of microbial populations in
    groundwater and, more recently, deep subsurface
    environments (2000 ft) have been studied and
    revealed substantial colonization (see Module 6)
  • Total microscopic counts of bacteria in a
    pristine (uncontaminated) shallow aquifer range
    from about 100,000 to 10,000,000 per gram dry
    weight.
  • Viable counts range from essentially zero to
    10,000,000 per gram. The lower numbers found with
    viable counting methods may reflect our ignorance
    of the conditions required for growth of the
    organisms.
  • In deeper aquifers, the situation is more
    variable some deep aquifer layers have almost no
    microorganisms while others have viable bacteria
    up to 100,000,000 per gram. Many of these
    bacteria seem to be growing under low nutrient
    levels they have morphologies and cell sizes
    typical of "starved" organisms.
  • Biomass measurements using ATP and membrane
    lipid determinations correlate reasonably with
    direct microscopic counts. Activity measurements
    based on respiration, metabolism of substrates
    are low but significant.

21
Overview of Microbiology 2
  • The types of bacteria present vary with depth.
  • The diversity of aquifer microbial populations is
    lower than at the surface in soils, but does not
    seem to decrease significantly with increasing
    depth.
  • Twenty-four genera were found by Hirsch et al
    (In Progress in Hydrochemistry. pp 311-325,
    Springer-Verlag, Heidelberg). The genera
    included
  • Pseudomonas
  • Achromobacter
  • Acinetobacter
  • Aeromonas
  • Alcaligenes
  • Chromobacterium
  • Flavobacterium
  • Moraxella
  • Caulobacter
  • Hyphomicrobium
  • Sphaerotilus
  • Gallionella
  • Arthrobacter
  • Bacillus
  • Gram negative bacteria predominated in sandy
    aquifers.
  • Filamentous bacteria and spores have only rarely
    been seen.

22
Sampling Techniques
Sampling Techniques
  • Soil sampling
  • Sampling of shallow layers of soil is performed
    using standard sampling methods for soils. Deeper
    samples of the soil and vadose zone can sometimes
    be taken by digging deeper pits and sampling
    horizontally with borers or sampling tubes.
  • Groundwater Sampling for Microbiological Assays.
  • There are many problems associated with sampling
    groundwater for microbiological purposes. One of
    the main problems is that of ensuring
    representative samples of both the groundwater
    and the mineral matrix of the aquifer.
  • Bacteria often adhere to particles and may not
    be equally distributed between the groundwater
    and the particles.
  • The groundwater is also moving through the
    matrix at various rates depending upon the
    permeability of the matrix and this can
    complicate sampling techniques.
  • The preferred method is to take core samples
    whenever possible so that both water and matrix
    material are disturbed as little as possible. In
    groundwater environments close to the surface
    (high water table), piston-driven cores can be
    forced into the aquifer material to obtain
    samples.
  • A hammer drill or similar device is used to
    force the sampling tubes (cores) into the soil
    and aquifer material. In cohesive matrix
    materials, withdrawing the core also withdraws
    the material and the tube can be stored until
    sampled.

23
Sampling Groundwater 1
  • The (usually aluminum) cores can be stored
    refrigerated until sampled, and the tubes can be
    easily sectioned into smaller lengths.
  • The outer peripheral layer of material that
    could have been in contact with the tube (and
    therefore could be contaminated) is not used.
  • Samples are taken from the interior of the core.
  • These can be diluted and plated or examined
    microscopically after staining (usually with
    fluorescent staining methods).
  •                                              

24
Sampling Groundwater 2
  • If groundwater samples are required from various
    depths in a drilled well, many different lengths
    of Teflon tube (an inert material) can be
    inserted into the well after it has been drilled
    or bored into the aquifer.
  • Many different techniques are available to drill
    the well, but these multi-level piezometer wells
    rely on being able to withdraw the well casing,
    leaving the tubes in place with their openings at
    different depths in the aquifer. Usually, many
    wells are drilled in an area to obtain a good
    coverage of the groundwater system.

25
Sampling Groundwater 3
  • Groundwater can be withdrawn from the tubes as
    required to obtain a complete three-dimensional
    "sample" of the groundwater in the area sampled.
  • One sampling system consists of a vacuum- or
    pump-driven, switchable manifold that allows the
    different tubes to be sampled and the water drawn
    into sterile sample bottles.

26
Detailed Sampling Distribution
Detailed Sampling Systems
  • One study Barbaro, Albrechtsen, Jensen,
    Mayfield and Barker Geomicrobiology Journal Vol
    12 203-219 (1995) has examined the distribution
    of bacteria over small distances in an aquifer
    (the aerobic zone of the Camp Borden aquifer).
  • The microbial numbers were determined for 9
    cores, 1.5 metres in length collected from the
    sand aquifer. They were from a zone that had not
    been used for other experiments, so it
    represented a "pristine" or normal condition.
  • Viable cell counts, electron transport system
    activity, dissolved oxygen levels, dissolved
    organic carbon levels and hydraulic conductivity
    were determined for contiguous samples at each
    10-cm interval in the cores.
  • The cores were arranged in a Y-shape with the
    open end of the "Y" facing towards the
    groundwater flow direction.
  • Maximum microbial occurrence and activity was at
    the top of the shallow aquifer and decreased
    rapidly with depth.
  • The activity was correlated with oxygen level
    and depth (these were also related, as might be
    expected).
  • Analysis also showed a correlation with
    dissolved organic carbon levels (these were low
    and only supported limited microbial growth)
  • Growth was stimulated only when a source of
    nitrogen was added.
  • This suggests that the limiting nutrient in the
    system was nitrogen. There was also a
    considerable difference between the various
    samples from similar depths in the 9 core samples
    and between contiguous samples in the same
    column.
  • This demonstrated the large degree of variation
    present in microbial distributions in this
    (relatively homogeneous) aquifer.

End of Section
27
Groundwater Microbiology Module 7a
- Groundwater Microbiology
  • General Overview of Groundwater (7)
  • Overview of Microbiology (7)
  • Sampling Methods (7)
  • Environmental Conditions in Groundwater
  • Contaminated Groundwater
  • Movement of Groundwater
  • Movement of Contaminants in Groundwater
  • Biodegradation and Kinetics
  • Groundwater Modeling

28
Chemical Physical Conditions
Chemical and Physical Conditions in Normal
Groundwater
  • The range of physical and chemical properties in
    groundwater environments can vary widely.
  • The particular properties will be dependent upon
    such things as
  • the origin of the rock materials forming the
    matrix
  • the chemical composition of materials earlier in
    the flow path
  • the state of the fracturing or the weathering of
    the materials
  • the grain size distribution
  • the age of the formation (how long it has been
    eluted by groundwater)
  • The nutrient status of groundwater varies widely
    according to the variation in physical and
    chemical properties.
  • To support microbial activities, the following
    must be present
  • carbon source for biomass production (and often
    energy production - by heterotrophic bacteria)

29
Element Composition
In terms of percentage dry weights, typical
cellular values for various elements are
These values should be present in the same
approximate concentrations in any environment
that allows microbial growth. As microbial
growth occurs, oxygen depletion may occur and
anaerobic conditions will be produced. This will
most likely occur under conditions of high
nutrient loading entering the groundwater from
any source. Typical situations are where a
landfill site leaches materials into the
groundwater, or where organic materials enters
from a contaminated or high nutrient status
surface water body. Since oxygen diffuses
10,000 times more slowly in water than in air,
and is sparingly soluble in water (typical values
are in the low mg/L range), microbial activity
can quite readily remove all available oxygen.
This will cause a change in the Eh or pE (redox
potential) of the groundwater. This redox
potential will then be further changed (assuming
oxygen is absent), by the presence and chemical
equilibria of the ions dissolved in the water.
Typically, growth in uncontaminated groundwater
is limited by the low carbon and/or nitrogen
levels present. The system is often C or N
limited. Addition of these elements in an
available form leads to increased microbial
activity. This activity can then lead to oxygen
depletion and production of anaerobic conditions
and low pE values (measured in millivolts)
30
Gasoline Spill
  • The number of contaminants entering groundwater
    is potentially very large.
  • Any source of contamination that can enter
    groundwater through surface waters, disposal
    practices, septic systems, air transport,
    run-off, infiltration from streams, lakes or
    rivers can lead to effects on groundwater.
  • Gasoline is a common contaminant of groundwater
    and forms a plume of soluble gasoline components
    in groundwater systems..

The hydrocarbons of gasoline float on the surface
and can move. The soluble components
dissolve in the groundwater and migrate
31
Advection
Movement of materials in groundwater 
Groundwater moves at slow rates depending on the
porosity and hydraulic conductivity of the
medium, through which it flows. Any compounds
dissolved in the groundwater also move, but the
movement is complicated by the adsorptive
processes of the compounds on the mineral and
organic parts of the rock material in the
aquifer. If the compound is not adsorbed at all
(chloride or bromide ions are examples) then it
moves at the same velocity as the water. This
is ADVECTION.
ANIMATION OF ADVECTION PROCESS
Use the Back command on your browser to
return to this page
Note that the water moves at the same speed as
the advected material (chloride ions in this
example)
32
Retardation
If the compound is adsorbed onto materials in the
matrix, the effective movement will be slower
than that of the groundwater this is
RETARDATION. Compounds that dissolve in lipids
also tend to more soluble in the organic matter
in soils and the groundwater matrix material.
The sorption distribution
coefficient (Kd) of the compound is a measure
of the degree of retardation that can be
expected. ANIMATION OF
CONTAMINANT MOVEMENT AND RETARDATION Use the
Back command on your browser to return to this
page
Note that the retarded compound (the red "plug")
moves at a slower rate than the water (the blue
arrow). It is RETARDED compared to the speed and
extent of water flow. This behaviour occurs
because the retarded compound is adsorbed onto
organic materials in the aquifer and then
released. This effectively "slows down" the speed
at which the material can move.
33
Dispersion
If the compound is undergoing biological or
chemical degradation as it travels, its actual
volume may become smaller as the materials is
used. Normally, however, without degradation,
the volume of the material becomes larger (but
less concentrated) as it moves because of the
processes of DISPERSION and DIFFUSION  
ANIMATION OF DISPERSION
PROCESS ANIMATION OF
BIODEGRADATION AND DISPERSION OF TOLUENE Use
the Back command on your browser to return to
this page
Note that the yellow "plug" of material is moving
at the same speed as the water (the blue arrow),
but that it is getting larger as it moves through
the aquifer matrix. This is because it is
"dispersing" or "diffusing"  as it travels.
34
Movement of Contaminants
Movement of contaminants
These processes can be seen in the movement of
"slugs" of toluene and chloride through an
aquifer. The toluene "slug" is being rapidly
degraded as it moves and is becoming smaller as
it moves slowly through the aquifer.
Dispersion, diffusion and
retardation can also be examined at a much
smaller scale that of the mineral grains and
organic materials in the groundwater matrix or
soil Animation of
advection, retardation and diffusion in an
aquifer matrix Use the Back command on your
browser to return to this page
 If, on the other hand, the compound IS adsorbed
(retarded) and IS biodegraded (such as toluene) -
then the shape of the slug of compound will be
very different. Even though dispersion is
occurring, it may be masked by the actual
disappearance of mass   due to biodegradation and
the "slowing" of the rate of transport of the
slug due to retardation (by adsorption)
35
Borden Experiment
A real experiment
In an experiment designed to show the fate of
gasoline soluble components in groundwater,
CHLORIDE, BENZENE and TOLUENE were injected into
the site at Camp Borden through an injection
well. The three-dimensional movement of the
resulting plume in the moving groundwater was
followed. The plan below shows the position and
extent of the "slugs" of chloride, benzene and
toluene (from left to right) after 3, 53 and 108
days. The slug at each of the three times is on
the same diagram. They are separated on the
diagram even though in the actual experiment they
were all moving along the same flow path. In
fact, no material was left at the injection wells
at 53 and 108 days. The contour lines in each
slug show the calculated three dimensional
concentration of the materials reduced to 2
dimensions for plotting. The data for these plots
was gathered from 20 depth samples at each of the
sampling wells on the diagram as the "slugs"
passed. The X and Y coordinates are in metres.
Groundwater movement direction
See animation on next slide
36
Animation - Borden
Animation of movement through the Borden
Aquifer Use the Back command on your browser
to return to this page
  • In the animation note that
  • The CHLORIDE ions move by a process of advection
    and dispersion/diffusion. Note that the chloride
    plume grows larger but less concentrated. There
    is no MASS LOSS but rather a movement and
    dispersion of the material through a larger
    volume. This kind of movement is typical of a
    CONSERVATIVE TRACER no biodegradation, no
    retardation, simply advection and dispersion with
    no mass loss.
  • Both the BENZENE and TOLUENE are retarded (they
    move a smaller distance than the chloride ions).
    There is very little mass change in the benzene
    but there is a large change in the toluene. The
    toluene is being biodegraded and has essentially
    disappeared by day 108.
  • Note the spreading of the materials in the
    linear direction of movement. This is due to the
    fact that dispersion in the direction of flow is
    usually greater than dispersion in other
    directions (see the plume for chloride and
    benzene at day 53)
  • Note that the plumes of different materials are
    not uniform in concentration throughout their
    volume. Differences in dispersion rates due to
    the heterogeneous nature of the aquifer materials
    leads to this uneven distribution of
    concentration (see the plume for chloride at day
    53).

37
Rates of Biodegradation
Rates of Biodegradation of Organic Compounds in
Various Environments in Groundwater
  • One of the major concerns when examining the
    biodegradation of chemicals in groundwater is the
    rate of the processes.
  • This is because of the fact that the movement of
    groundwater leads to migration of materials that
    can then cause problems at remote sites.
  • This becomes a legal issue of responsibility for
    clean-up of those contaminants. The only "safe"
    situation is where the plume does not migrate
    past the property boundary of the company or
    person causing the pollution. It is then a matter
    of cleaning up that site and preventing any
    migration to another property.
  • The issues of rate of biodegradation and
    microbial activity are obviously closely linked.
    It is important to establish which reactions are
    possible or probable and how fast they are likely
    to occur in a particular contaminated groundwater
    system.

38
Effect of Environment
The environment has a very large effect on rates
of biodegradation anaerobic versus aerobic
degradation rates are widely different in most
cases.
Increasing anaerobic conditions
A typical groundwater contaminated with organic
material (e.g.. leachate from a landfill site)
shows a series of different zones over a distance
in the flow path.
39
Redox Reactions
  • Redox Reactions
  • Redox reactions are the most common type of
    reaction, modifying or removing compounds from
    groundwater environments.
  • The groundwater environment often develops
    different redox potentials due to growth of
    microorganisms and consequent removal of oxygen
    followed by a progressive reduction in Eh or pE
    due to the growth and activities of other
    microorganisms (see evolution of groundwater).
  • The response of different groups of
    microorganisms to chemical contaminants at
    different redox conditions is an important aspect
    of biodegradation.
  • This response can be divided into two parts
  • Energy Yield (Thermodynamic equations)
  • Rates of Degradation (Kinetics)
  • These are NOT the same a reaction can be
    thermodynamically possible and actually yield
    energy, but it is so slow without the presence of
    "catalytic" enzymes from bacteria (for instance),
    that it will not be significant in the
    environment.

40
Energy Yield
  • Energy Yield (Thermodynamics)
  • Every redox reaction consists of two half
    reactions - an oxidation and a reduction. In
    theory, any set of half reactions can be combined
    and an energy yield calculated this does NOT
    mean that pairs of half reactions that yield
    energy will necessarily be fast enough to be
    significant, only that at equilibrium the energy
    yield will be "X" kcals.
  • The speed at which the reactions reach
    equilibrium is a function of the kinetic, not the
    thermodynamic, equations.
  • One way to examine these half reactions is to
    look at a series of oxidation and reduction
    reactions and calculate the energy yields by
    pairing them. If the energy yield is positive,
    then the reaction is thermodynamically possible
    without the input of external energy - i.e. it is
    an energy-yielding reaction. If growth is to
    occur, then energy yielding reactions are
    required.
  • We can calculate Free Energy for various half
    reactions important in groundwater environments
    and present the results as a "delta G DG in
    kilocalories per mole of reactants". That is the
    change in free energy in kilocalories per mole.
  • These half reactions can then be combined to
    calculate (algebraically) the delta G for the
    combined reactions. This will provide the energy
    yield for that set of reactions. 

41
Example
For example
42
Half Reactions
It is now possible to combine the half reactions
to calculate the energy yields from the various
reactions  
43
Series Reactions
Reactions in Series It is also possible to use
the same concepts and calculations to examine the
situation where a series of reactions occur in
sequence (leading to a series of intermediates
that are then metabolised to other compounds).
Each step in the process can be assigned a set
of reactions that will, in total, sum to give the
overall reaction of the entire process. An
example is the process of denitrification
occurring with methanol as the substrate Step
1. 0.067 CH3OH 0.2 NO3- 0.067 CO2 1.33
H2O 0.2 NO2- (nitrate to nitrite) Step 2.
0.100 CH3OH 0.2 NO2- 0.2 H 0.1 CO2 0.3
H2O 0.1 N2 (nitrite to nitrogen gas)
Overall 0.157 CH3OH 0.2 NO2- 0.2 H 0.1
CO2 0.3 H2O 0.1 N2 (denitrification of
nitrate to nitrogen gas)
44
Overview
Overview If this general process is carried
out for the various combinations of organic
substrates and electron acceptors, the following
summary graph is obtained
45
Summary of Figure
  • From the previous Figure
  • Greater energy is represented by greater
    negative values (-22 means more energy release
    than -10)
  • Nitrite is the most efficient electron acceptor
    - more efficient than oxygen.
  • There is decreasing energy availability for ALL
    electron donors as the electron acceptor changes
    from nitrite to oxygen to nitrate to sulfate to
    carbon dioxide (in that order).
  • The compounds listed as electron donors (methane
    to formate) are typical compounds found in
    organic matter entering groundwater or produced
    in situ in groundwater with high organic carbon
    input.
  • They are typical metabolic products of microbial
    activity in groundwater.
  • Combination of electron donors with any electron
    acceptor leads to increasing energy yields from
    methane to acetate to benzoate to succinate to
    ethanol to lactate to glycine to pyruvate to
    methanol to glycerol to glucose to formate (in
    that order).

46
Groundwater Evolution
  • Relationships to Groundwater Evolution Process
  •  The order of decreasing energy availability is
    the same as the order of biochemical reactions
    observed in a groundwater plume.
  • The most "available" or "utilised" electron
    acceptors are those that yield the highest energy
    per mole under the particular environmental and
    Eh conditions.
  • Removal of oxygen leads to utilisation of
    nitrate as electron acceptor.
  • Removal of nitrate leads to utilisation of
    sulfate as electron acceptor.
  • Removal of sulfate leads to utilisation of
    carbon dioxide as electron acceptor.
  • Not all organic compounds are utilised as
    electron donors under all conditions.
  • Some are only utilised by certain groups of
    bacteria.

47
Overview
Overview If this general process is carried
out for the various combinations of organic
substrates and electron acceptors, the following
summary graph is obtained
48
Kinetics
49
Kinetics 1
Kinetics
The kinetics of biodegradation are a set of
empirically derived rate laws. Three suffice to
describe most biological reactions dCA/dt
-k0 Zero order dCB/dt -klCA First order
dCB/dt -k2CACB Second order k0, k1, k2
rate constants mol/1-sec, /sec, 1/mol-sec,
respectively CA, CB some reacting species
This can be applied to the reaction of the
compounds with a surface such as a metal
catalyst, a soil surface or an enzyme. Two
extremes of concentration can be delineated the
first is when there are few molecules of reactant
(CA) and many of the surface. In this case, few
of the available sites will be covered, so the
reaction rate dCA/dt is proportional to the
concentration of A (first order reaction above).
Secondly, when CA is so large that every site
is saturated with A, the rate is constant (zero
order reaction above). The combined function of
these reactions can be written 
Where k' ko/kl
50
Kinetics 2
This is the very common biological form of the
equation for growth on a substrate as the
concentration of the substrate is increased. It
leads to Michaelis-Menton (or Monod-) type
kinetics. The saturation coefficient (Ks) is
the concentration of   substrate equal to half
that causing saturation of the enzyme sites (zero
order). It is that same as adsorption onto a
surface-area-limited substrate. The enzyme
sites or the adsorbing sites are "saturated".
The enzyme cannot operate faster, and the
adsorbing substrate cannot adsorb any more
material. Bacterial growth kinetics are
slightly more complex and follow the classical
"Monod-type" kinetics. In this case, the rate
of substrate utilisation is proportional to the
concentration of the microorganisms present X
and is a function of the substrate concentration.
The Monod bacterial growth kinetics are
traditionally written as
Where S substrate concentration k
maximum utilisation rate for the substrate per
unit mass of bacteria X concentration of
bacteria Ks half-velocity coefficient for
the substrate y yield coefficient dX/dS
51
Kinetics 3
OR, in graphical terms
Ks values typically range from 0.1 to 10.0 mg/L.
Groundwater systems therefore usually operate
in the range where Ks is more than S. In this
particular case, the equation reduces to second
order kinetics 
52
Kinetics 4
  • If substrate concentrations are low, the reaction
    becomes first order with respect to both
    substrate and bacterial population size. This has
    been confirmed experimentally in many sites and
    with many systems.
  • There are really three kinds of kinetic models
    used in describing biotransformations in soils
    and groundwater systems.
  • The first, BATCH model kinetics, are those
    described above. They deal with the utilisation
    and biotransformation of the substrate and the
    growth of bacteria over time in a closed system.
  • The second, CONTINUOUS model kinetics deal with a
    more-or-less constant flow of the substrate
    through or into a known volume system. These
    models are useful for predicting results of slow
    but continuous processes.
  • The third is that of BIOFILM model kinetics.  It
    is  based on the theory that the bacteria are
    attached to solid particles in the subsurface
    environment and behave accordingly.
  • This last model still uses Monod-type kinetics
    but extends the model to include the effects' of
    biofilm thickness and diffusion of substrate into
    and out of the biofilm. More than likely the
    actual "biofilms" in the field situation are so
    sparse as to simply constitute a random
    distribution of individual cells attached to
    mineral or organic matter particles. They cannot
    be considered ''biofilms'' in the engineering
    sense.
  • In particular, subsurface environments where the
    substrate content and concentration is very high
    (landfill site leachates ?), some degree of
    biofilm may be present, but calculations of
    population densities and actual direct
    observations should always be done to confirm
    this possibility.

53
Kinetic Models
Based on the log concentration of substrate log
S and the log of thee biomass log B. It is
possible to predict what kind of kinetic model
should apply
54
Kinetic Models 2
Same graph as the previous slide now in
non-logarithmic plot of Substrate remaining
versus Time
55
Cometabolism 1
Where does co-metabolism fit into these kinetic
models ? Co-metabolism and Secondary Substrate
Utilization There are a number of compounds in
the environment which are transformed by
microorganisms, yet it has been difficult or
impossible to find organisms that can use them as
a source of carbon and/or energy. The compounds
may be transformed sequentially by a series of
bacteria or other microorganisms such that no
organisms gained energy sufficient to allow
growth or cell division, from the reactions  It
is necessary to have an alternate or primary
substrate for growth under these conditions. A
good definition of this co-metabolism is " the
transformation of a non-growth substrate in the
obligate presence of a growth substrate or
another transformable compound'.
Some examples
56
Cometabolism 3
A more comprehensive example comes from the work
of Dalton Stirling (1982) who examined the
enzyme methane monooxygenase (MMO). This enzyme
catalyzes the NAD(P)H-driven insertion of oxygen
into a wide variety of compounds such as
n-alkanes. haloalkanes, alkenes, ethers and
aromatic, alicyclic and heterocyclic compounds.
They found that with MMO, of 31 compounds
oxidized, 5 were only oxidized by resting cells
and 7 were oxidized only in the presence of 4mM
formaldehyde. None of the compounds were able
to support growth and replication at the normal
growth temperature in a period of 10 days. 
57
Cometabolism 4
   
58
Contaminated Groundwater 1
  • The most common contaminants found in groundwater
    are derived from activities involving production
    or use of synthetic organic compounds such as
    organic solvents, pesticide and other chemical
    production facilities, fuels and fuel additives.
    dye production, plastics production and use, and
    various chemical feedstock operations. In
    addition, plants such as wood treatment plants
    can introduce metal contamination (arsenic and
    copper), oil refineries can introduce metal
    catalyst residues, and disposal practices can
    introduce many other metal forms.
  • Generally, these chemicals are introduced during
    production, transport, storage, utilisation, as
    feedstocks in other processes or loss by
    dispersion during use, spillage, accident and
    improper disposal.
  • The most common organic contaminants are
  • chloroform
  • trichloroethylene
  • carbon tetrachloride
  • tetrachloroethylene
  • 1,1,1-trichloroethane
  • dichloroethylenes
  • dibromochloropropane
  • methylene chloride
  • and, in lower amounts
  • toluene
  • benzene
  • xylene

59
Contaminated Groundwater 2
The most commonly found pesticides are herbicides
(typically alachlor, 2,4-D and atrazine) and soil
fumigants or sterilants (such as
1,2-dichloropropane and EDB). This reflects the
heavier use patterns for these compounds compared
to the insecticide group of pesticides. The
presence of metal ions in groundwater is
dependent on the pE and pH of the environment.
The solubility of elements such as aluminum,
manganese, iron, cobalt, and others depends very
much on the pH of the system. For instance,
aluminum is much more soluble at lower, acidic,
pH levels. The redox state of the environment
determines to a large extent the valance state of
the element in solution.
60
Groundwater Flow Path 1
Summary of Microbial Activities and Environmental
Conditions in Groundwater Flow Path
  • Conditions in the flow path of groundwater after
    the introduction of available organic materials
    follow a distinct evolutionary process the order
    is from
  • aerobic activity to
  • heterotrophic anaerobic activity to
  • denitrification activity to
  • sulfate reduction activity to
  • methanogenic activity.
  • A typical example would be under a landfill site
    leaching small quantities of organic matter into
    the groundwater.
  • If massive amounts of leachate are entering the
    groundwater system, the evolutionary development
    path for the different "zones" in the groundwater
    occur very quickly and would be much closer
    together. In extreme cases, the entire range of
    denitrifying, sulfate-reducing and methanogenic
    activity occurs with the landfill itself.
  • The consequence of this is the production of
    significant quantities of methane gas.
  •  
  • There are many reasons, all linked, why these
    processes occur in this order. They can be
    considered separately but are in fact closely
    related to one another.
  •  

61
Groundwater Flow Path 2
1. Development of anaerobic conditions (removal
of oxygen) and then progressively more reducing
conditions (below).
62
Groundwater Flow Path 3
2. Development of bacterial groups because of
their tolerance or lack of tolerance of oxygen
and other nutrients in the system (below).
63
Groundwater Flow Path 4
3. Development of specific groups of bacteria
based on the carbon sources remaining in the
plume after previous bacterial activities (below)

64
Groundwater Flow Path 5
4. Development of different groups of bacteria
based on the relative energy yields from the
organic carbon source with specific electron
acceptors (oxygen, nitrate, sulfate and carbon
dioxide) (below).
65
Interactions
  • INTERACTIONS
  • The reducing conditions are produced by the
    bacteria progressively using up all the available
    oxygen, which cannot be replaced quickly because
    of the slow diffusion of oxygen in water.
  • More reducing conditions are then produced by
    bacteria that use different electron acceptors in
    the "chain". These same bacteria are adapted to
    thrive under those particular redox conditions.
    They use the available substrates until they are
    exhausted when another group of bacteria (the
    next in the chain) then starts to use the
    remaining substrates.
  • As the substrates are utilised by the bacteria,
    the remaining substrates yield less and less
    energy with the available electron acceptors

66
Groundwater Modelling 1
Groundwater Modelling
  • There are two aspects to groundwater modelling
    that are of interest to environmental
    microbiologists.
  • The modelling of groundwater flow and
    environmental conditions in aquifers
  • Modelling the activities (including
    biodegradative) of microorganisms in groundwater.
  • When the two are linked, it will be possible to
    predict the fate and transport of contaminants in
    groundwater systems. This is not an easy set of
    problems to solve, nor is it easy to get the data
    and information required to construct robust
    models of either of these two main areas of
    interest.
  • Modelling of groundwater flow and environmental
    conditions in aquifers.
  • According to Freeze and Cherry (1979) in their
    book "Groundwater", there are 4 main steps in
    modelling groundwater
  • 1. Examination of the physical problem
  • 2. Replacement of the physical problem by an
    equivalent mathematical problem
  • 3. Solution of the mathematical problems using
    accepted mathematical techniques

67
Groundwater Modelling 2
The overall view of this is that the main
difficulty in modelling (of ALL types) is a
problem of interpretation and interconversion
between "real" physical problems and the
mathematical interpretations of those problems.
This is compounded by a lack of data and
understanding about certain aspects of the
problems (e.g. the actual kinetic rates of
biodegradation in field conditions). The
processes that have to be considered in physical
models of GROUNDWATER FLOW and ENVIRONMENTAL
CONDITIONS are
 These can be combined into a model that can be
used to predict results. Note that
microbiological parameters are included even in
the model for GROUNDWATER FLOW and ENVIRONMENTAL
CONDITIONS. This is because the biological and
biochemical activities of microorganisms affects
the conditions in the aquifer (see REDOX effects
in previous lectures). Thus, even though we
have arbitrarily separated the two types of
models (environmental conditions/groundwater flow
and microbiological activity), in fact they are
closely linked.
68
Groundwater Modelling
2. Modelling the activities (including
biodegradative) of microorganisms in groundwater
Example Model An example model will be used
to demonstrate the concepts involved. It is a
simplified model in that it does not deal with
all of the parameters listed above. It is a
realistic model in that it does deal with a real
physical problem. The site is Camp Borden,
Ontario - but it is extended to predict results
in other types of aquifers. The problem addressed
is groundwater flow and oxygen concentrations in
flow regimes in that aquifer. The
characteristics used for the physical nature of
the groundwater sediment is based on Borden sand,
a silty sand and a coarse sand. The properties of
these are
 When the model for BTEX movement and oxygen
concentration in the various aquifer materials is
run, the following results were obtained
(Sudicky, et al. Earth Sciences, University of
Waterloo). Animation of benzene-oxygen
relationships at Camp Borden Aquifer Use the
Back command on your browser to return to this
page
69
Key Points
  • Key Points
  • Groundwater Introduction Environment
  • Hydrologic cycle and its importance to
    groundwater
  • Concept of aquifer and groundwater flow
  • Confined and unconfined aquifers
  • Recharge and discharge zones
  • Water table and hydraulic head
  • Hydraulic conductivity
  • Groundwater Microbiology
  • General types of bacteria present in groundwater
    environments
  • Sampling problems and processes for water and
    matrix material
  • Microbial Processes
  • Chemical and physical processes in normal
    groundwater
  • C and N limitation
  • Movement of groundwater
  • Movement of contaminants by advection,
    dispersion, retardation and biodegradation.
    Effects on observed distances of movement

70
Key Points 2
  • Kinetics
  • Definitions of zero order, first order and
    second order kinetics
  • Types of responses obtained under those
    different types of kinetics when organic
    materials are biodegraded in groundwater
  • Mechanisms causing variation in  Groundwater
    plume conditions
  • Overview of the microbial mechanisms responsible
    for the observed conditions in a groundwater flow
    path contaminated with organics 
  • The four main reasons why the processes occur in
    the order observed.
  • Groundwater Modelling
  • Parameters of importance in groundwater
    modelling - physical and biochemical/chemical
  • Summary and Integration
  • Factors to be considered in groundwater studies
    of contaminated groundwater (and normal
    groundwater) environments
  • Geochemical nature of the volume of groundwater
  • Bioenergetics of the processes
  • Biotransformations or biodegradation processes
  • Site conditions

End of Module
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