BemidjiPaper2

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Progression of natural attenuation processes

at a crude oil spill site:

II. Controls on spatial distribution of

microbial populations

Barbara A. Bekins a,*, Isabelle M. Cozzarelli b, E. Michael Godsy a,Ean Warren a, Hedeff I. Essaid a, Mary Ellen Tuccillo c

aUS Geological Survey, MS 496, 345 Middlefield Road, Menlo Park, CA 94025, USAbUS Geological Survey, 431 National Center, 12201 Sunrise Valley Drive, Reston, VA 20192, USA

cDepartment of Environmental Sciences, Clark Hall, University of Virginia, Charlottesville, VA 22903, USA

Received 7 March 2000; received in revised form 25 January 2001; accepted 13 February 2001

Abstract

A multidisciplinary study of a crude-oil contaminated aquifer shows that the distribution of

microbial physiologic types is strongly controlled by the aquifer properties and crude oil location.

The microbial populations of four physiologic types were analyzed together with permeability, pore-

water chemistry, nonaqueous oil content, and extractable sediment iron. Microbial data from three

vertical profiles through the anaerobic portion of the contaminated aquifer clearly show areas that

have progressed from iron-reduction to methanogenesis. These locations contain lower numbers of

iron reducers, and increased numbers of fermenters with detectable methanogens. Methanogenic

conditions exist both in the area contaminated by nonaqueous oil and also below the oil where high

hydrocarbon concentrations correspond to local increases in aquifer permeability. The resultsindicate that high contaminant flux either from local dissolution or by advective transport plays a key

role in determining which areas first become methanogenic. Other factors besides flux that are

important include the sediment Fe(II) content and proximity to the water table. In locations near a

seasonally oscillating water table, methanogenic conditions exist only below the lowest typical water

table elevation. During 20 years since the oil spill occurred, a laterally continuous methanogenic

zone has developed along a narrow horizon extending from the source area to 50 60 m

downgradient. A companion paper [J. Contam. Hydrol. 53, 369386] documents how the growth of

the methanogenic zone results in expansion of the aquifer volume contaminated with the highest

0169-7722/01/$ - see front matterD 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 9 - 7 7 2 2 ( 0 1 ) 0 0 1 7 5 - 9

* Corresponding author. Fax: +1-650-329-4463.

E-mail address:[email protected] (B.A. Bekins).

www.elsevier.com/locate/jconhyd

Journal of Contaminant Hydrology 53 (2001) 387406


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concentrations of benzene, toluene, ethylbenzene, and xylenes. D 2001 Elsevier Science B.V. All

rights reserved.

Keywords: Anaerobic environment; Microorganisms; Contaminant plumes; Ground water; BTEX; Biodegrada-tion

1. Introduction

The establishment of aquifer microbial populations that degrade organic ground-water

contaminants is a key aspect to the success of natural attenuation of these compounds.

Studies of the degradative capabilities of aquifer microorganisms have documented spatial

variability in both degradation rates (Adrian et al., 1994; Nielsen and Christensen, 1994)

and ability to degrade specific compounds (Anderson et al., 1998). Such variability ispresumably due a number of underlying causal factors. These include variations in the

physical and chemical properties of aquifer materials, and in the aqueous concentrations of

contaminants and other important reactants (NRC, 1993). Significant progress in address-

ing these issues has been made by focusing on each aspect separately. For example, Webb

and Anderson (1996) used modeling to show how variations in aquifer physical properties

causes preferential contaminant migration along connected high permeability pathways. In

contaminated aquifers where the geochemical conditions have been exceptionally well

characterized, it has been noted that contaminant concentrations and associated geo-

chemical conditions change over sub-meter scales (Cozzarelli et al., 1999). Moreover,

studies of microbial populations attached to the sediments in contaminant plumes indicate

that microbial physiologic types also vary on sub-meter scales (Bekins et al., 1999; Smith

et al., 1991).

Although it is clear that geochemical conditions and microbial physiologic zones can

vary on small spatial scales, studies are needed that relate this variation to the subsurface

chemical and physical properties. The results of these studies provide insight into how

spatial and temporal changes in aquifer conditions leads to a succession of microbial

physiologic types. This is important because the succession of microbial physiologic types

is associated with a change in the degradative capabilities (e.g. Krumholz et al., 1996). In

some cases, microorganisms with specific degradative capabilities are found in favorablehabitats that exist in only a portion of a plume (e.g. Anderson et al., 1998). Methods that

provide a basis for identifying specific habitats allow the capabilities of the associated

microbial populations to be characterized. An increased understanding of how redox zones

and associated microbial capabilities change over time facilitate forecasts of the long-term

fate of contaminant plumes.

This study is part of a larger effort to investigate the long-term fate of a plume of crude-

oil contaminants. The goal of this work was to examine the processes that control the

spatial distribution of microbial physiologic types. Using the most probable number

(MPN) method, the microbial population distributions were estimated for four physiologic

types: aerobes, iron reducers, heterotrophic fermenters, and methanogens. In the samelocations, pore-water chemistry, nonaqueous oil saturation, sediment iron content, and

sediment permeability were also determined. The combined data set presented in this study

B.A. Bekins et al. / Journal of Contaminant Hydrology 53 (2001) 387406388


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provides insight into controls on the patterns of microbial ecological succession at the

plume scale. These results can be related to observations of the long-term evolution of the

plume described in a companion paper by Cozzarelli et al. (2001) to show how the

microbial population succession has affected the contaminant plume.

2. Site description

The study aquifer, located near Bemidji, MN (Fig. 1), is a surficial formation of pitted

and dissected glacial outwash sediments. The sediments of the unconfined aquifer consist

of moderate to poorly sorted sandy gravel, gravelly sand, and sand with thin interbeds of

fine sand and silt (Franzi, 1988). Dillard et al. (1997) examined the permeability (k, in

square meters) using grain-size analyses and the Krumbein and Monk (1942) relation. The

Fig. 1. Map of the Bemidji research site showing the oil pipeline, location of the 1979 pipeline break, nonaqueous

oil body know as the north pool, water table contours, and locations of sample wells.

B.A. Bekins et al. / Journal of Contaminant Hydrology 53 (2001) 387406 389


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overall mean and variance of the log(k) distribution were 11.28 and 0.86, respectively.

The distribution was bimodal with 11% low permeability silty material and the remainder

higher permeability coarse-grained material. The water table is 6 10 m below land surface

and the direction of ground-water flow is to the northeast (Hult, 1984). The average linearvelocity of the groundwater is about 0.05 m/day in the silt layers and 0.150.5 m/day in

the coarser-grained sediment (Bennett et al., 1993).

The aquifer was contaminated with crude oil when a pipeline ruptured in August 1979.

After clean-up operations by the pipeline company, the unrecovered spilled oil infiltrated

into the aquifer. The oil is entrapped as a residual nonaqueous phase in the vadose zone

and also forms two bodies of oil floating on the water table (Dillard et al., 1997; Hult,

1984). The largest oil body (the north pool, Figs. 1 and 2) was estimated to contain

147,000 l of oil in 1998 (Herkelrath, 1999).

A set of papers (Baedecker et al., 1993; Bennett et al., 1993; Eganhouse et al., 1993)

described the geochemical evolution of the contaminant plume emanating from the north

pool. Starting in 1984, concentrations of both reduced iron and methane began to increase

in the anoxic zone immediately downgradient from the oil body. From 1986 to 1989,

profiles of dissolved organic compounds indicated the plume reached a quasi steady state

in which dissolution from the nonaqueous oil was approximately balanced by iron

reducing and methanogenic biotransformation (Baedecker et al., 1993; Lovley et al.,

1989). Fig. 2A and B contain contour plots of dissolved oxygen (DO) and dissolved

volatile organic carbon (VOC) measured in 1992 along the axis of the north pool plume

(AAV, Fig. 1) (Baedecker et al., 1993; Cozzarelli et al., 1996). Note that the anaerobic

core of the plume attains a minimum vertical thickness of about 1 m at the downgradientedge of the nonaqueous oil body. Essaid et al. (1995) showed that the plume thickens

vertically below the oil body as groundwater flow is diverted around the area where

partial saturation with oil decreases the hydraulic conductivity. Observations on the

continued chemical evolution of the plume since 1992 are presented by Cozzarelli et al.

(2001).

On the basis of the pore-water chemistry, Baedecker et al. (1993) divided the aquifer in

the vicinity of the oil body into anoxic, transition, and background zones. Within the

anoxic zone, the primary anaerobic degradation processes are iron reduction and methano-

genesis (Baedecker et al., 1993; Lovley et al., 1989). Bekins et al. (1999) used MPN data

from three vertical profiles and seven broadly spaced samples to map microbialphysiologic zones for aerobes, iron reducers, and methanogens in the aquifer near the

oil body (Fig. 3). Data from the three vertical profiles show that populations of aerobes,

iron reducers, fermenters, and total methanogens vary sharply over sub-meter scales. The

variations in microbial numbers are systematic indicating that different physiologic types

dominate in different portions of the contaminated aquifer.

Degradation of petroleum hydrocarbons under methanogenic conditions requires the

combined activity of fermenting bacteria that break down complex organics and two types

of methanogens (e.g. Schink, 1997). Locations in the Fig. 3 profiles where methanogens

increase together with fermenters indicate the presence of a methanogenic consortium

capable of utilizing petroleum hydrocarbons for growth. In the anoxic zone, there is also apositive correlation between numbers of aerobes and methanogens. This implies that the

organisms from the anoxic zone that grew in our media under aerobic conditions are



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capable of functioning both aerobically and anaerobically. In locations where these

aerobes increase with methanogens, the aerobes most likely function as fermenters in

a methanogenic consortium (Bekins et al., 1999). In the profiles, the numbers of bacteria

functioning as fermenters exceed those of methanogens by factors of 10104. Such ratios

are comparable to those found in other studies of methanogenic consortia both in the

laboratory (e.g. Morgan et al., 1991) and in the field (Godsy et al., 1992). Thus, areas ofthe aquifer classified as methanogenic contain culturable methanogens together with

increased numbers of fermenters or aerobes and decreased numbers of iron reducers.

Fig. 2. Cross-sections of 1992 concentration distributions in the contaminant plume below the north pool oil body

along the line AAVof Fig. 1. The area with greater than 10% oil saturation determined by Dillard et al. (1997) is

also shown. (A) Dissolved oxygen distribution modified from Cozzarelli et al. (1996) together with the microbialsample locations. The double lines mark the locations of detailed vertical profiles A, B and C. (B) Total dissolved

volatile organic carbon are from Baedecker et al. (1996). Screened intervals and ID numbers of monitoring wells

are also shown.



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A correlation analysis of the combined MPN data set showed an inverse correlation

between iron reducers and methanogens indicating that methanogenic conditions arepresent in areas where iron reduction is less favorable. Methanogenic zones have evolved

in two places: one within the area with separate-phase oil and a second below the oil in the

Fig. 3. Vertical MPN profiles and interpreted cross-section showing the distribution of microbial physiologic

types in the anaerobic portion of the Bemidji plume. The vertical profiles correspond to Sites A, B and C in Fig. 2.

The interpreted cross-section is based on the vertical profile data combined with data for the point samples

marked on the plot (Bekins et al., 1999).



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laterally migrating plume of contaminated ground water. In the laterally migrating plume,

there is a classic pattern of a methanogenic core progressing with depth to iron-reducing

and then aerobic conditions. Moreover, the establishment of methanogenic conditions

appears more advanced near the oil body at Sites A and B, than downgradient at Site C.This is evident from the higher peak methanogen numbers and the lower minimal values

of iron reducers at Sites A and B compared to Site C. The anaerobic core of the plume

attains a minimum vertical thickness of about one meter at the downgradient edge of the

nonaqueous oil body (Site B, Fig. 2) (Cozzarelli et al., 1996). The microbial data from this

location indicate that the methanogenic zone centered at 422.5 m is especially narrow,

spanning a distance of only 25 cm, in contrast to the thicker 0.51 m methanogenic zones

at Sites A and C.

3. Methods

On the basis of the existing geochemical data, locations for three vertical profiles in the

anaerobic portion of the plume (Sites A, B and C; Fig. 2A) were chosen to characterize the

microbial population, pore-water chemistry, sediment iron content, and sediment perme-

ability. In September of 1996, two cores with a horizontal separation of 1 m were collected

from Site B (cores 9609 and 9610). Each core was 2.3 m long, which was sufficient to

span the entire vertical extent of the anaerobic plume at this location. One core was

analyzed for the microbial population on the sediments, along with extractable iron [Fe(III)

and Fe(II)], and grain size. The second core was used for pore-water chemical analyses.Because the drill rig depth measurements were imprecise, data from the two cores were

aligned vertically using the grain-size distributions. In August of 1997, five additional

cores were collected. Three cores were needed to span a vertical interval from the oil body

to the base of the contaminant plume at Site A (cores 97049706), and two cores were

sufficient to obtain a vertical profile of the plume at Site C (cores 97019702). In these

cores, the pore water was drained for chemical analyses and the sediments were analyzed

for microbial numbers, grain-size distributions, and extractable iron.

The cores were collected with a freezing drive shoe that freezes the bottom 10 cm of the

core in situ before retrieval thus preserving the position of the ground water with respect to

the cored sediments (Murphy and Herkelrath, 1996). The 47-mm diameter cores werecollected in clear polycarbonate liners pre-rinsed with methanol and de-ionized water. For

pore-water chemical analyses, water was drained from the core at 15-cm intervals. The pH

was measured in the field and the water samples were preserved for laboratory analyses of

hydrocarbons, dissolved organic carbon, methane, and major cations, according to the

methods described by Cozzarelli et al. (2001). The sediment grain-size analyses were

performed using the method described by Hess et al. (1992). Permeabilities were estimated

from sediment grain-size distributions using the method of Krumbein and Monk (1942).

The sediment iron was determined by 0.5 M HCl extraction as described by Cozzarelli et

al. (2001). This method was expected to extract the bioavailable Fe(III) as well as poorly

crystalline Fe(II) from the sediments.Sediment samples were collected at 2070 cm intervals and analyzed for populations

of aerobes, iron reducers, heterotrophic fermenters, and methanogens using the MPN



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method. The methods of sediment sampling and MPN determination are described in

detail by Bekins et al. (1999). Briefly, approximately 10 g of sediment from the center

of the core was collected under a flow of oxygen-free nitrogen gas and added to a pre-

reduced and anaerobically sterilized (PRAS) mineral salts solution containing a non-ionic surfactant added to detach microbes from the sediment (Yoon and Rosson, 1990).

The tube was then sealed, mixed well, and allowed to stand for 2 h to allow pene-

tration of surfactant into the sample. The tubes were then opened and sonicated to

dislodge the bacteria into the mineral salts under a flow of sterile oxygen-free nitrogen

gas.

Microbial concentrations were determined using a five-tube MPN analysis. Samples

were serially diluted by orders of magnitude into PRAS mineral salts solutions. Bacteria

capable of aerobic heterotrophic growth were enumerated using Standard Methods Broth

(BBL Microbiology Systems, Cockeysville, MD). Tubes with visible growth after

incubation at room temperature for one week were scored positive. Microorganisms

capable of anaerobic heterotrophic growth or fermentation were enumerated using PRAS

prepared Schaedlers Broth (Difco, Detroit, MI). Tubes with turbid growth or clumps of

particulates after incubation at room temperature for 1 week were scored positive.

Iron-reducing bacteria were enumerated using PRAS prepared media consisting of

sodium acetate and poorly crystalline iron in a mineral salts solution (modified from

Lovley and Phillips, 1986). After inoculation, the serum bottles were aseptically pres-

surized with a 70:30 mix of H2/CO2 to 140 kPa and then incubated for a minimum

of 6 weeks at room temperature. A serum bottle was scored as a positive if greater

than 2 mg/l of reduced iron (Fe

2+

) was present as determined with bipyridine. Micro-organisms capable of methane production were enumerated on PRAS mineral salts

media. Acetoclastic and formate-utilizing organisms were enumerated under a nitrogen

and CO2 atmosphere with the addition of acetate or formate, respectively. Hydrogen

oxidizers were enumerated by aseptically pressurizing the serum bottles after inoculation

with a 70:30 mix of H2/CO2 to 140 kPa. The serum bottles were allowed to incubate for

a minimum of 6 weeks at room temperature. The presence of methane in the head space

was determined by Gas Chromatography/Flame Ionization Detection analysis (Godsy,

1980). Methanogenic numbers presented in this paper represent the sum of the numbers

obtained for the three types of methanogens at a given location. Although this will result

in double counting some methanogens, this method provides the best indication of themaximum spatial extent of methanogenic activity. Results from this study of the

enumerations for the separate physiologic types of methanogens are published in Warren

et al. (1999).

4. Results and discussion

We now consider how the physical and chemical conditions in the aquifer have affected

the locations of the physiologic zones. Fig. 4 shows plots for Sites A, B and C of the iron-

reducer and methanogen MPNs together with the extractable sediment Fe(II) and Fe(III),pore-water concentrations of ethylbenzene and the sum of m- and p-xylene, sediment

permeabilities, and pore-water Fe(II) concentrations.



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Fig. 4. Vertical profiles for Sites A, B and C of microbial data for iron reducers and methanogens with interpreted

methanogenic zones of Fig. 3 shown as shaded gray bands. Plots of Fe(II) and Fe(III) concentrations from the

sediments; pore-water concentrations of ethylbenzene and m- and p-xylene; and permeabilities estimated from

sediment grain-size distributions and pore-water Fe(II) concentrations for each site are shown on the same vertical

scale. Uncontaminated background concentrations of sediment Fe(II) and Fe(III) are shown in the sediment iron

plots, averaging 6 and 25 mmol/g, respectively.



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4.1. Extractable iron on sediments

In general, extractable concentrations of sediment Fe(III) are lower, and Fe(II) are

higher in the area of the aquifer affected by the plume than background values.Sediment Fe(III) values at the base of the core at Sites B and C match background

concentrations indicating that the profiles span the entire anoxic portion of the plume.

The overall picture is consistent with the results of Tuccillo et al. (1999) who found

that microbial iron reduction of the petroleum hydrocarbons has resulted in depletion of

solid phase Fe(III) and precipitation of Fe(II) minerals on the sediments in the anoxic

zone. However, the microbial numbers and sediment iron concentrations together

provide a more comprehensive picture of the processes controlling the progression

from iron reduction to methanogenesis. First, in the methanogenic zones, extractable

iron and culturable iron reducers are still present. Moreover, detailed examination of

the sediment iron and microbial data shows that high concentrations of solid-phase

Fe(III) do not always correlate with high numbers of iron reducers and vice versa.

Below is a detailed discussion of these issues focusing on Sites A and B because these

sites have been anoxic the longest and have the greatest variability in sediment Fe

concentrations.

In areas with culturable methanogens there are also culturable iron reducers and

significant extractable Fe(III) suggesting ongoing low levels of iron reduction. The lowest

value of extractable Fe(III) found in all of the cores is 11 mmol/g which is almost 50% of

the average background concentration. Apparently, only about half of the Fe(III) extracted

on the aquifer sediments is used before methanogenesis begins. The sediment ironextraction method recovers primarily ferrihydrite and only a small percentage of Fe(III)

from crystalline phases such as hematite, goethite and magnetite (Tuccillo et al., 1999). A

statistically significant inverse correlation between iron-reducers and methanogens

(Bekins et al., 1999), suggests that there is a continuum of decreasing iron-reducing

activity in proportion to the increase in methanogenic activity. This observation is

consistent with a range of iron phases in the aquifer that vary in their bioavailability.

Postma (1993) showed that simultaneous sulfate and iron reduction is thermodynamically

feasible if microorganisms are forced to use less soluble iron phases than the poorly

crystalline material that typically comprises hydroxypolymer coatings on mineral grains.

The Fig. 4 data suggest that a similar analysis is needed to determine whether simulta-neous iron-reduction and methanogenesis is feasible under the geochemical conditions in

the contaminated portion of the Bemidji aquifer.

High numbers of iron reducers correspond to some of the lowest sediment Fe(III)

concentrations, at three locations within the oil body and just below it (Site A, 424 m and

Site B, 423.4 and 422.8 m). Preferential use of favorable Fe(III) phases, along with natural

variability of the Fe(III) phases on the sediments, may play a role. However, the proximity

of these locations to the separate-phase oil appears to be significant. There are several

mechanisms that may facilitate use of less-favorable iron phases near the separate-phase

oil. One possibility is that proximity to the oil provides access to more favorable substrates

such as toluene oro-xylene. Another important aspect is that organic ligands are present inhigher concentrations near the oil (Cozzarelli et al., 1994). Lovley et al. (1994)

demonstrated that organic ligands facilitate chelation of iron from solid Fe(III) phases.



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Alternatively, the organic ligands may bind to aqueous Fe(II) reducing the effect of

product inhibition (Urritia et al., 1999).

Very low numbers of iron reducers correspond to moderate levels of Fe(III) in three

locations (Site A, 423.2 m and Site B, 422.4 and 422.9 m). These locations also have peakvalues of sediment Fe(II) and active methanogens. The data suggest that high concen-

trations of sediment Fe(II) can inhibit iron reduction and provide an advantage to the

methanogens. Inhibition of iron reduction due to coating of Fe(III) oxides by precipitated

Fe(II) surface phases has been documented in the laboratory (Roden and Zachara, 1996;

Urritia et al., 1998). Cozzarelli et al. (1999) also noted at a gasoline-contaminated aquifer

that coating of iron oxide surfaces by reduced-iron phases apparently limited iron

reduction and allowed sulfate reduction to become more energetically favorable.

4.2. Flux of hydrocarbon contaminants

To illustrate the relationship between the flux of organic contaminants through the

aquifer and the microbial population distribution, the concentrations of ethylbenzene and

m- andp-xylene for the three sites are shown in Fig. 4. Ethylbenzene was selected because

it persists like benzene in the anoxic plume (Cozzarelli et al., 1990) but, unlike benzene, is

present at concentrations similar to m- and p-xylene. The behavior ofm- and p-xylene is

intermediate compared to toluene, which degrades rapidly, and benzene, which degrades

more slowly (Eganhouse et al., 1993). Between Sites B and C, the average concentration

of ethylbenzene drops slightly from about 0.4 to 0.3 mg/l. In contrast, the concentration of

m- and p-xylene drops from 12 mg/l at Sites A and B to about 0.1 mg/l at Site C.At Sites A and B, methanogenic conditions occur in two distinct zones in the anoxic

portion of the aquifer (gray shaded zones in Fig. 4). The upper zone corresponds to the

portion of the aquifer where separate-phase oil is present. The lower zone corresponds to

the center of the horizontally advecting plume below the oil. Both of these methanogenic

zones are associated with higher concentrations of ethylbenzene, m- and p-xylene, and

dissolved iron. The correspondence between methanogenic zones and higher concen-

trations of contaminants suggests that over time these areas have been subjected to greater

cumulative hydrocarbon fluxes.

In the zones where separate-phase oil is present at Site A (above 423 m) and Site B

(above 422.8 m), peak iron reducer numbers are lower than the maximum values found inthe aquifer and methanogens are present in all samples (Fig. 4). In this area, partial oil

saturations exceeding 10% reduce the hydraulic conductivity thus restricting downward

advection of hydrocarbons through the oil (Essaid et al., 1995). Eganhouse et al. (1996)

showed that the concentration of hydrocarbons in contact with the separate-phase oil is

controlled by equilibrium dissolution. Thus, although the advective flux of hydrocarbon-

contaminated water is low, the flux due to dissolution of the separate-phase oil is relatively

high in this zone.

Hydrocarbon flux in the plume below the oil occurs primarily by horizontal advection

of water contaminated by separate-phase oil located upgradient. At Sites A and B, a

methanogenic zone has developed in the center of the plume where the maximumadvective BTEX flux occurs. To illustrate this point, Fig. 5 shows the methanogen

numbers together with the computed flux of benzene, toluene, ethylbenzene and xylenes



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(BTEX) for the three vertical profiles. The computed flux is the product of the average

water table gradient between Sites A and C (0.0024), the estimated permeabilities, and the

total BTEX concentrations. Values between data points for BTEX and permeability were

estimated by linear interpolation. The correspondence between increased flux andmethanogens numbers is generally quite good. The higher peak flux at Site B is associated

with a higher number of methanogens compared to Site A. Unfortunately, the peak in flux

near the base of Site A (419.7 m) cannot be associated with a peak in methanogens

because no microbial data exist for this location. A Pearson correlation analysis for the 14

methanogen MPN data values from below the oil body together with the computed BTEX

fluxes at the corresponding elevations gives a significant value of 0.53 (n =14; p < 0.06).

In areas of lower flux, found along the plume fringes and in low permeability zones,

iron-reducer numbers remain high. In these locations, the relatively slow migration of

contaminated water by transverse dispersion and diffusive flux results in a lower

cumulative hydrocarbon flux over time. Thus, the available Fe(III) is less depleted andthese areas still support high populations of iron-reducers compared to nearby locations

with higher hydrocarbon flux. As a consequence, the plume under the oil body still

contains both methanogenic and iron-reducing zones even though this portion of the

aquifer has been anaerobic for many years.

At Site C, the picture is more complex because the location of peak flux is above the

methanogenic zone in the top 50 cm of the saturated zone (Fig. 5c). To understand these

data, we examined the water level changes in well 531 located adjacent to Site C (see Fig.

2 for locations). Between 1985 and the date of the microbial analyses in 1997, the water

levels fluctuated about 0.6 m seasonally. The shaded area in Fig. 5c shows the average

water table over this period plus or minus two standard deviations. Culturable metha-nogens are found only below the lowest level of the water table. Presumably, the seasonal

presence of oxygen makes locations above the annual low water table toxic to metha-

Fig. 5. Computed flux of total BTEX plotted together with methanogenic and iron-reducing MPN data for the

three vertical profiles in Fig. 3. For profiles (A) and (B), only the intervals below the oil are plotted. Over these

intervals, there is a significant positive correlation between total BTEX flux and methanogen numbers. For profile

(C), water table elevations fluctuate seasonally over the shaded gray region and methanogens are present only

below the lowest water table elevation.



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nogens. Another indication that changing redox conditions affect the microbial data at this

location is the coexistence of iron reducers and aerobes at both the top and base of the

profile. Dissolved Fe(II) is below detection and the cores were visually more orange-

colored at both the top and the base of the profile compared to the center. Theseobservations suggest that dissolved oxygen is intermittently present at the fringes of the

profile creating conditions that alternately favor each population.

The effects of water table oscillations on the Site C profile also obscure the relationship

of hydrocarbon flux to the microbial zones. During water table low stands, the advective

flux of contaminants through elevations above the water table is negligible whereas the

flux through the positions at or below the water table continues to occur. The 1997 data

correspond to a year in which the water table reached a level of 423.84 m, which is higher

than any value measured in previous years. If flux were adjusted for relative time during

which the water levels lie above the minimum water table, the peak in the effective

hydrocarbon flux plot would lie well below that shown. However, this type of analysis is

not possible with the existing data because seasonal variations in profiles of pore-water

chemistry are not available.

4.3. Modeling of the biogeochemical processes

Essaid et al. (1995) constructed a two-dimensional solute transport model with

biodegradation for the Bemidji, MI, crude oil spill site. The simulations included the

sequential degradation of volatile and nonvolatile fractions of dissolved organic carbon by

aerobic processes, manganese reduction, iron reduction, and methanogenesis. Despite theconsiderable uncertainty in the model parameter estimates, results of simulations repro-

duced the general features of the observed ground-water plume and the measured

microbial concentrations from the time of the spill in 1979 to 1992. To examine the

predicted long-term behavior of the plume and microbial populations, the simulation of

Essaid et al. (1995) was extended from 13 to 20 years.

Fig. 6 shows the simulated concentrations at a location 36 m downgradient from the

center of the oil body for volatile and nonvolatile dissolved organic carbon (VDOC and

NVDOC, respectively); solid phase Mn(IV); solid phase Fe(III); and aerobic, manganese /

iron reducer, and methanogenic biomass. It is interesting to note that the simulations

predict that the VDOC concentration (this includes benzene, toluene, ethylbenzene,m-,p-ando-xylene) reaches a minimum at about 11 years following the spill and then increases.

The increase in VDOC concentration corresponds to the depletion of solid phase Fe(III).

The increase in VDOC concentrations as Fe(III) is depleted is in agreement with recent

observations at the site (Cozzarelli et al., 2001).

The manganese/iron (Mn/Fe) reducer biomass concentration shows a rapid increase

during the first 5 years following the spill when manganese reduction is the dominant

process. As Mn(IV) is depleted, the biomass concentration decreases slightly until iron

reduction becomes significant, resulting in increased Mn/Fe reducer growth. The Mn/Fe

reducer biomass reaches a peak at about 12 years and then begins to decrease as

Fe(III) is depleted. The biomass of the methanogenic consortium in the simulationsbegins to dominate the population after 16 years. It is important to note that the model

combines the activities of the entire methanogenic consortium into one process. Thus,



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Fig. 6. Simulated concentrations at a location 36 m downgradient from the center of the oil body for (a) volatile

and nonvolatile dissolved organic carbon (VDOC and NVDOC, respectively), (b) solid phase manganese (Mn4+),

solid phase iron (Fe3+), and (c) aerobic, manganese/iron reducer, and methanogen biomass. Simulated

concentrations of VDOC rise slowly between 15 and 20 years as solid phase iron oxides are depleted.



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the overall degradation rate of hydrocarbons to carbon dioxide and methane represents

the rate-limiting step in combined series of fermentation and methanogenesis reactions.

Moreover, the methanogenic biomass in the model reflects the biomass of the total

consortium. The growth of the methanogenic consortium in the simulations does notappear to be limited by nutrients during the simulated time period. In reality, although

the supply of carbon for growth is plentiful, other required nutrients such as

phosphorus or nitrogen will limit the growth of the methanogens and fermenters in

the aquifer.

The prediction by the simulations that the biomass of the methanogenic consortium will

eventually exceed that of iron reducers correctly reflects the character of the microbial data

in the designated methanogenic zones at sites A and B (Figs. 3 and 4). These two sites are

both located upgradient of the simulated location. The microbial data show that the aquifer

at site C has not yet reached the stage where the methanogenic consortium dominates the

population. Similarly, this site is located downgradient of the simulated location. The

modeling indicates that the methanogenic zone expands downgradient at an average rate of

2.25 m/year. However, this prediction is based on a homogeneous aquifer with average

properties for the Bemidji site. The microbial data indicate that after almost 20 years the

populations of iron reducers and the methanogenic consortium at Site C are almost equal.

Thus, the data from the three vertical profiles together with the widely spaced sample

locations shown in Fig. 3 indicate that a horizontally continuous methanogenic zone

extends downgradient for almost 60 m from the center of the oil body (Bekins et al.,

1999). Thus, the actual rate of horizontal expansion of the methanogenic zone is almost 3

m/year.The plots in Figs. 4 and 5 suggest that the methanogenic zone occurs along a

connected, high flux pathway in the Bemidji aquifer. This observation is consistent with

previous modeling results of contaminant plumes by Webb and Anderson (1996). They

showed that in subsurface systems with contrasts in hydraulic conductivity, such as

glacial outwash aquifers, the location and magnitude of flow is constrained by the

internal sediment structures. A recent paper by Harvey and Gorelick (2000) argues that

solute transport in a heterogeneous aquifer can be described by advection through

mobile regions together with rate-limited diffusion into small-scale immobile regions.

Our data suggest that this model accurately describes solute transport and biodegrada-

tion at the Bemidji site and explains some aspects of the observed microbial distribution.Within the advecting plume, methanogenic zones evolve first in the mobile regions

where the hydrocarbon flux is highest. The higher flux of contaminants causes a more

rapid decrease in sediment Fe(III) oxides allowing methanogenic conditions to develop

more rapidly in these regions than a strictly homogeneous aquifer model would predict.

In contrast, iron-reducing conditions persist longer than the homogeneous model

predicts in the immobile regions of the aquifer where diffusive mass transfer is rate

limiting.

Aqueous phase electron acceptors may also be depleted first in longitudinally

contiguous high flux pathways. Scholl et al. (1999) demonstrated that dissolved electron

acceptors in an initially uncontaminated aquifer are depleted fastest in high permeabilityzones. Because longitudinal dispersivity is usually 100 times greater than transverse

dispersivity (Garabedian et al., 1991), the plume preferentially mixes with uncontami-



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nated water in the principle direction of flow. Thus, the plume grows longitudinally as

electron acceptors are depleted from the water and sediments along the high permeability

horizons. In contrast, lower permeability immobile regions retain supplies of favorable

electron acceptors longer because the biodegradation rate is limited by diffusion ofcontaminants into and electron acceptors out of the immobile regions.

4.4. Implications for monitored natural attenuation

Although high flux zones where methanogenic conditions develop are laterally

continuous, they may be very thin vertically, making them difficult to locate. Cozzarelli

et al. (1999) noted that very thin redox zones at a gasoline-contaminated site resulted

in mixed geochemical indicators in well-water samples. At Site C, the methanogenic

niche spans an interval of


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little degradation through the portion of the aquifer depleted in Fe(III) but degraded

rapidly in the zone where significant Fe(III) is still present. At the Bemidji site,

Geobacterspp. capable of oxidizing benzene under iron-reducing conditions are found

only in the zone where significant Fe(III) is present (Anderson et al., 1998; Rooney-Varga et al., 1999) and benzene degrades rapidly in this zone (Anderson and Lovley,

1999).

5. Summary and conclusions

Combined studies of microbial populations together with physical and chemical

conditions in the Bemidji aquifer have provided insights into the long-term behavior of

petroleum hydrocarbon plumes. The dominant physiologic type of the microbial

population changes from iron reducing to methanogenic over vertical scales as small

as 25 cm. Near the oil body, methanogenic zones have evolved in two high-flux areas.

One zone is found within the nonaqueous oil where high fluxes are due to local

dissolution and diffusion of contaminants from the oil. A second zone exists along high

permeability horizons in the laterally migrating contaminant plume. At a distance of 65

m downgradient from the oil, lower numbers of methanogens indicate that methanogenic

conditions are less advanced than below the oil. The spatial correspondence between

methanogenic zones and peaks in hydrocarbon flux suggest that the methanogenic zone

in the plume is growing laterally along a narrow, connected, high permeability pathway.

Results presented by Cozzarelli et al. (2001) show that the growth of the methanogeniczone results in a slow expansion of the hydrocarbon plume. A computer simulation of

the plume predicted an initial decrease in concentrations between 5 and 10 years after

the spill at an observation well 36 m downgradient of the oil body, followed by an

increase in simulated concentrations between 10 and 15 years as iron oxides are depleted

and iron-reducing bacteria decrease in number. At sites with large petroleum hydro-

carbon plumes from persistent nonaqueous sources, risk analyses should account for

expansion of methanogenic conditions along narrow, connected high permeability

pathways.

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