v

CONTENTS

 Abstract ..................................................................................................................................................................................1

Introduction ............................................................................................................................................................................1

Purpose and scope .........................................................................................................................................................5

Description of study area...............................................................................................................................................5

Methods and data analysis......................................................................................................................................................6

Microbial community analysis ......................................................................................................................................6

Biodegradation data analysis .........................................................................................................................................8

Assessment of wetland microbial communities .....................................................................................................................8

Assessment of biodegradation of chlorinated volatile organic compounds.........................................................................12

Summary and conclusions....................................................................................................................................................13

References cited ...................................................................................................................................................................15

Appendix–Bacterial profiles ................................................................................................................................................16

A1. Bacterial terminal-restriction fragment length polymorphism profiles (TRFLP) in sediment from

Lauderick Creek surficial sediment collection sites, December 18, 2001 ........................................................17

A2. Bacterial terminal-restriction fragment length polymorphism profiles (TRFLP) in sediment from

Lauderick Creek surficial sediment collection sites, April 9, 2002 ..................................................................18

A3. Bacterial terminal-restriction fragment length polymorphism profiles (TRFLP) in sediment from

Lauderick Creek surficial sediment collection sites, June 18, 2002 .................................................................19

Figures

1. Map showing location of the Cluster 13, Lauderick Creek, and West Branch Canal Creek areas,

Aberdeen Proving Ground, Maryland ....................................................................................................................2

2. Map showing concentration of total volatile organic compounds in ground water at Cluster 13,

Lauderick Creek area..............................................................................................................................................3

3. Map showing well locations and wetland areas at Cluster 13, Lauderick Creek area................................................4

4. Map showing U.S. Geological Survey wetland sediment sampling locations in the

Lauderick Creek area and selected previous ground-water sampling sites ............................................................7

5. Graphs showing percent peak areas [area of peak on terminal-restriction fragment length

polymorphism (TRFLP) profile divided by the total area of all peaks on the profile] for

three bacterial peaks [represented by their base pair (bp) size] that are believed to be

associated with 1,1,2,2-tetrachloroethane (TeCA) biodegradation and were observed in

wetland sediment samples collected at (A) West Branch Canal Creek, winter 1999 and

(B) Lauderick Creek, winter 2001..........................................................................................................................9

6. Graphs showing percent peak areas [area of peak on terminal-restriction fragment length

polymorphism (TRFLP) profile divided by the total area of all peaks on the profile] of

bacterial peaks [represented by their base pair (bp) size] (A) 89/90 bp and (B) 172 bp 

for wetland sediment samples collected at Lauderick Creek in winter (December 2001)

and summer (June 2002).......................................................................................................................................10

7. Graphs showing distribution of 1,1,2,2-tetrachloroethane (TeCA) and its anaerobic daughter

products in wetland porewater from (A) site PW-12, Lauderick Creek, October 1998,

and (B) site WB26, West Branch Canal Creek, May 1999 ..................................................................................12
vi

Figures—Continued

8. Graph showing estimated first-order 1,1,2,2-tetrachloroethane (TeCA) degradation rate

calculated using the natural log of TeCA concentrations (normalized by chloride

concentrations) at site PW-12, Lauderick Creek area, and estimated traveltime in

wetland porewater (calculated assuming vertical ground-water flow velocity of

0.9 meters per year) ..............................................................................................................................................13

Tables

1. Location and background information on wetland sediment grab samples collected in the

Cluster 13, Lauderick Creek area..............................................................................................................................6

2. Detections of dehalorespiring bacteria in the Dehalococcoides and Desulfuromonas groups in

 wetland sediment grab samples collected in the Cluster 13, Lauderick Creek area, 2001–02 ..............................11

3. Abundance of Methanosarcinaceae as a percentage of the total methanogen community in

wetland sediment grab samples collected in the Cluster 13, Lauderick Creek area, 2001–02 ...............................11

4. Abundance of Methanosarcinaceae as a percentage of the total methanogen community in

wetland sediment grab samples collected in the West Branch Canal Creek area, March 1999..............................11

Conversion Factors, Vertical Datum, and Abbreviations

_____________________________________________________________________________________________________________________________________

Multiply By To obtain

_____________________________________________________________________________________________________________________________________

Length

centimeter (cm) 0.3937 inch

millimeter (mm) 0.03937 inch

meter (m) 3.281 foot

meter (m) 1.094 yard

Volume

liter (L) 33.82 ounce, fluid

liter (L) 2.113 pint

liter (L) 1.057 quart

liter (L) 0.2642 gallon

liter (L) 61.02 cubic inch

Flow rate

meter per year (m/yr) 3.281 foot per year

_____________________________________________________________________________________________________________________________________

Temperature in degrees Celsius ( °C) may be converted to degrees Fahrenheit ( °F) as follows:

°

F = (1.8 x °C ) + 32

Concentrations of chemical constituents in water are given either in micrograms per liter (μg/L)

or micromoles per liter (μmol/L).
Abstract 1

Preliminary Assessment of Microbial Communities and

Biodegradation of Chlorinated Volatile Organic Compounds 

in Wetlands at Cluster 13, Lauderick Creek Area, 

Aberdeen Proving Ground, Maryland

By Michelle M. Lorah, Mary A. Voytek, and Tracey A. Spencer

Abstract

A preliminary assessment of the microbial 

communities and biodegradation processes for 

chlorinated volatile organic compounds was conducted by the U.S. Geological Survey in wetlands 

at the Cluster 13, Lauderick Creek area at 

Aberdeen Proving Ground, Maryland. The U.S. 

Geological Survey collected wetland sediment 

samples from 11 sites in the Lauderick Creek area 

for microbial analyses, and used existing data to 

evaluate biodegradation processes and rates. The 

bacterial and methanogen communities in the 

Lauderick Creek wetland sediments were similar 

to those observed in a previous U.S. Geological 

Survey study at the West Branch Canal Creek wetland area, Aberdeen Proving Ground. Evaluation 

of the degradation rate of 1,1,2,2-tetrachloroethane 

and the daughter compounds produced also 

showed similar results for the two wetlands. However, a vertical profile of contaminant concentrations in the wetlands was available at only one site 

in the Lauderick Creek area, and flow velocities in 

the wetland sediment are unknown. To better 

evaluate natural attenuation processes and rates in 

the wetland sediments at Lauderick Creek, chemical and hydrologic measurements are needed 

along ground-water flowpaths in the wetland at 

additional sites and during different seasons. Natural attenuation in the wetlands, enhanced bioremediation, and constructed wetlands could be 

feasible remediation methods for the chlorinated 

volatile organic compounds discharging in the 

Lauderick Creek area. The similarities in the 

microbial communities and biodegradation processes at the Lauderick Creek and West Branch 

Canal Creek areas indicate that enhanced bioremediation techniques currently being developed for 

the West Branch Canal Creek wetland area would 

be transferable to this area.

Introduction

Cluster 13 of the Lauderick Creek area, Aberdeen 

Proving Ground (APG), Maryland was used for various 

chemical warfare-related training activities from 1920 until 

the 1950s (General Physics Corporation, July 1999) (fig. 1). 

Although historical records are incomplete, it is known that 

decontamination of chemical warfare material and training 

activities on clothing impregnation took place. One of the 

common military decontaminants and components of clothing-impregnating solutions was 1,1,2,2-tetrachloroethane 

(TeCA) (Nemeth, 1989; Lorah and others, 1997). The 

major volatile organic compounds (VOCs) detected in 

ground water in the Cluster 13, Lauderick Creek area are 

TeCA and trichloroethene (TCE), which are present at maximum concentrations of 86,000 and 6,000 μg/L (micrograms 

per liter), respectively (General Physics Corporation, July 

1999) (fig. 2). Ground-water contamination extends to wetland and creek-bottom areas along Lauderick Creek, especially along the south tributary (figs. 2 and 3). 

Since 1992, the U.S. Geological Survey (USGS) has 

studied natural attenuation of VOCs, primarily TeCA and 

TCE, in a freshwater tidal wetland along West Branch Canal 

Creek, which is southwest of Lauderick Creek in the Edgewood area of APG (Lorah and others, 1997; Lorah and Olsen 

1999a, 1999b; Lorah and others, 2003) (fig. 1). Field and 

laboratory data showed that rapid anaerobic biodegradation 

is a major process in the wetland sediments at the West 

Branch Canal Creek wetland area and that monitored natural 

attenuation could be an effective ground-water remediation 

method at this site. In these wetland sediments, TeCA is 

biodegraded by simultaneous dichloroelimination and 

hydrogenolysis reactions, resulting in the production of 

1,2-dichloroethene (both cis and trans isomers) (12DCE) 

and 1,1,2-trichloroethane (112TCA), respectively. These 

intermediate compounds biodegrade to vinyl chloride (VC) 

and 1,2-dichloroethane (12DCA), and ultimately to non- 
2 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD
Introduction 3
4 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD
Introduction 5

chlorinated, non-toxic VOCs (Lorah and others, 1997). 

Abiotic degradation of TeCA to TCE also occurs, but this 

reaction accounted for less than 10 percent of the TeCA 

removed in laboratory experiments (Lorah and others, 1997; 

Lorah and others, 2003). In 1999, the USGS began an 

intensive study of the microbial communities associated with 

biodegradation reactions in the wetland sediments at the 

West Branch Canal Creek study area (Lorah and others, 

2003). A microbial consortium, rather than one individual 

microbial species or group, is involved in the degradation of 

TeCA and TCE in the wetland sediments at West Branch 

Canal Creek (Lorah and others, 2003). Critical microbial 

species that are involved in the degradation of TeCA and 

TCE have been tentatively identified by obtaining terminalrestriction fragment length polymorphism (TRFLP) profiles 

of the bacterial and methanogen communities during microcosm experiments with the wetland sediment (Lorah and 

others, 2003). This TRFLP technique is commonly referred 

to as DNA fingerprinting. Molecular probes also were used 

to determine the presence of the anaerobic dehalorespiring 

bacteria Dehalococcoides ethenogenes and Desulfuromonas

species in the wetland sediment at some sites in the West 

Branch Canal Creek wetland area. These dehalorespiring 

bacteria can use chlorinated VOCs as terminal electron 

acceptors to derive energy and grow, allowing more rapid 

biodegradation of the VOCs than through cometabolic 

reduction reactions (Maymó-Gatell and others, 1997, 2001).

The microbial communities in the wetland sediments at 

the Cluster 13, Lauderick Creek area have not been characterized previously, although wetland porewater sampling has 

been conducted to determine the distribution of TeCA and 

TCE degradation products and to characterize the redox conditions (General Physics Corporation, July 1999). From 

October 2001 through September 2002, the USGS, in cooperation with the Environmental Conservation and Restoration Division at APG, conducted a preliminary investigation 

in the wetlands at Cluster 13, Lauderick Creek area. The 

objectives of this study were to characterize the microbial 

communities present in the surficial wetland sediments at the 

Cluster 13, Lauderick Creek area, and to conduct a preliminary evaluation of the technical feasibility of using natural 

attenuation or enhanced bioremediation as a remediation 

method for the ground-water plume. Microbial communities in the Cluster 13 wetland area were compared to available USGS data from the West Branch wetland study area to 

evaluate the presence or absence of critical microbial species 

involved in TeCA degradation. This evaluation was conducted to determine the validity of using available data on 

degradation pathways and rates from the West Branch wetland area (Lorah and others, 2003) to evaluate wetland remediation methods at Lauderick Creek. In addition to the use 

of monitored natural attenuation in the wetlands as a groundwater remediation method, ongoing studies at the West 

Branch wetland area are focused on developing methods to 

enhance in situ bioremediation of VOCs in “hotspots,” 

including ground-water seeps. Possible enhancements 

include the addition of nutrients or electron donors, or the 

addition of critical microbial species (bioaugmentation) to 

the existing wetland sediments. The preliminary characterization of the microbial communities and biodegradation 

rates in the Lauderick Creek wetland area presented in this 

report was used to evaluate the transferability of the ongoing 

West Branch wetland remediation studies to Lauderick 

Creek.

Purpose and Scope

The purpose of this report is to (1) present the results of 

microbial community analyses of surficial wetland sediment 

samples from the Cluster 13, Lauderick Creek area, and (2) 

provide a preliminary assessment of the chlorinated VOC 

biodegradation processes and rates in the wetlands of the 

Lauderick Creek area using the collected microbial community data and existing ground-water data for this area. The 

microbial community and biodegradation in wetlands of the 

Lauderick Creek and West Branch Canal Creek areas are 

compared. Sediment samples for microbial community 

analyses were collected three times from the Lauderick 

Creek area (December 2001, April 2002, and June 2002). 

Microbial community analyses for the Lauderick Creek wetland sediments are compared to similar analyses conducted 

for the West Branch Canal Creek wetland area during 1999–

2000 (Lorah and others, 2003). An initial evaluation of the 

biodegradation processes and potential for use of wetlands 

for ground-water remediation of VOCs in the Lauderick 

Creek area is given using available data for the Lauderick 

Creek area (General Physics Corporation, July 1999) and the 

West Branch Canal Creek area (Lorah and others, 1997; 

Lorah and others, 2003).

Description of Study Area

The Cluster 13, Lauderick Creek area is on a peninsula 

bordered by two tributaries (north and south tributaries) of 

Lauderick Creek, a tidal creek of the Bush River estuary (fig. 

2). The area consists of approximately 67 percent secondary growth upland forest, 25 percent open grassy fields, 

5 percent wetlands, and 3 percent developed areas (General 

Physics Corporation, July 1999). The wetlands primarily 

are along the northern sides of the north and south tributaries 

(fig. 3) and consist of freshwater (palustrine) forested wetlands, brackish (estuarine) intertidal emergent wetlands, and 

palustrine emergent wetlands (General Physics Corporation, 

July 1999). Wetland plants common to the palustrine emergent areas include phragmites (or common reed), cattails, 

and rushes, whereas the estuarine emergent species include 

cordgrass, three squares, and rushes (General Physics Corporation, November 1999).

A shallow surficial sand aquifer that is generally about 

6 m (meters) thick is contaminated with chlorinated VOCs 

(fig. 2). The surficial aquifer consists of silty sands and 

well-sorted sands in the upper section, and gravelly sands 

and gravels in the lower section (General Physics Corporation, July 1999). A thick clay unit and an uncontaminated 

lower confined aquifer underlie the surficial aquifer (General 

Physics Corporation, July 1999). Three possible source 

areas in the surficial aquifer have total VOC concentrations 
6 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD

that are indicative of the presence of dense non-aqueous 

phase liquids (DNAPLs) (areas greater than 10,000 μg/L in 

fig. 2). All three areas are along the northern side of the 

south tributary of Lauderick Creek, and wetlands are located 

immediately downgradient from two of the three source 

areas (figs. 2 and 3). Chlorinated VOC contamination, 

including TeCA, TCE, and their anaerobic daughter compounds (primarily 12DCE and VC), also exists in the shallow ground water in the wetland and south tributary (General 

Physics Corporation, July 1999). One surface-water sample 

collected offshore in the south tributary had a TeCA concentration of 72 μg/L (total VOCs of 100 μg/L) (General Physics Corporation, November 1999), and one ground-water 

seep sample collected on the wetland surface adjacent to the 

south tributary had a TeCA concentration of 41 μg/L (total 

VOCs of 70 μg/L) (General Physics Corporation, July 

1999).

Methods and Data Analysis

Microbial Community Analysis

Grab samples of the surficial wetland sediment 

were collected manually from 11 sites in the Cluster 13, 

Lauderick Creek area during three different seasons—winter 

(December 18, 2001), spring (April 9, 2002), and summer 

(June 18, 2002). Sediment sampling sites along the south 

tributary to Lauderick Creek were selected to cover a range 

of contaminant concentrations using available site data 

(General Physics Corporation, July 1999) (table 1; fig. 4). 

Only the south tributary wetlands were sampled because 

they are the most extensive and are in the most contaminated 

area in the Cluster 13, Lauderick Creek area. Sediment samples were placed in plastic bags and immediately frozen for 

later microbial analyses by TRFLP analysis on targeted gene 

products. Molecular analyses of the microbial communities were done in the USGS laboratory in Reston, Virginia.

Total genomic DNA was extracted from the sediment 

samples using the Bio101 FastDNA spin kit for soil according to manufacturer’s instructions (Bio101 Inc., Vista, California). The bacterial and methanogen communities in the 

samples were characterized using a polymerase chain reaction (PCR) procedure to amplify specific targeted DNA and 

performing TRFLP analysis on the PCR products (Clement 

and others, 1998). In order to evaluate bacterial communities, 16S rDNA was targeted for analysis because it codes for 

a molecule present in all prokaryotes and can differentiate 

individual species and groups. PCR was performed to 

amplify a portion of the bacterial 16S rDNA using the primers 46forward with a fluorescent tag (FAM) and 519reverse 

(Brunk and others, 1996) at a concentration of 0.2 μmol/L 

(micromoles per liter). To characterize the methanogenic 

community, a key functional gene involved in methanogenesis, mcrA, was amplified and analyzed. McrA DNA 

amplifications were performed using the degenerate mcrA 

oligonucleotide primer pair at 0.2 μmol/L (Klein and others, 

 

1988), except the forward primer was labeled with the fluorescent tag FAM. PCR conditions for bacterial and mcrA 

amplifications are given in Lorah and others (2003).

To obtain molecular fingerprints after amplification, bacterial 16S rDNA amplicons were digested overnight at 37 °C 

(degrees Celsius) with the restriction enzyme MnlI, whereas 

mcrA amplicons were digested with RsaI (New England 

Biolabs, Beverly, Massachusetts). Digests were precipitated 

in ethanol, centrifuged, dried, and resuspended in TrisEDTA buffer. Samples were electrophoresed on an ABI310 

sequencer (Applied Biosystems, Foster City, California), 

generating a plot of the base-pair size (using internal standards TAMRA 500) and the relative intensity of each fluorescent fragment. Undigested mcrA amplicons also were 

analyzed to determine the proportion of Methanosarcinaceae 

in the methanogen population on the basis of fragment 

length heterogeneity. The size of the PCR mcrA products 

differs between the Methanosarcinaceae (464 base pairs), the 

only group that contains acetotrophs, and the other main 

methanogen groups (481 base pairs)—Methanococcales, 

Methanobacteriales, and Methanomicrobiales. Because of 

this fragment length heterogeneity, the proportion of 

Methanosarcinaceae in the total methanogen community can 

be determined.

Table 1. Location and background information 

on wetland sediment grab samples

collected in the Cluster 13,

Lauderick Creek area

 [VOCs, volatile organic compounds; total concentrations in 

micrograms per liter given for adjacent well sites as reported by 

General Physics Corporation (July 1999); if the well site 

included several screened intervals, the highest concentration of 

total VOCs is given here. Different well types reported by 

General Physics Corporation (July 1999) included: WLC, 

monitoring well; PW, U.S. Geological Survey porewater 

sampler; PLC, Waterloo Multilevel Samplers]

Sediment

site number

Site number of

adjacent well

Total VOCs in

adjacent well 

SS1 WLC-25 1.7

SS2 PW-12 2,212

SS3 PW-11; PLC-01 1,904; No data

SS4 PW-10 3,311

SS5 WLC-28; WLC-82 1,335; 15

SS6A PLC-02 318

SS6B PW-07 106

SS7 PW-08 636

SS8 PW-08 636

SS9 PW-06; PLC-03 No data; 1,130

SS10 PW-05; PLC-03 1,117; 1,130
Methods and Data Analysis 7
8 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD

Each sediment sample also was amplified with primers 

(dhc 730f and dhc 1350r) specific for two groups of dehalorespiring bacteria—Dehalococcoides group (Dehalococcoides ethenogenes and Dehalococcoides sp. strain FL2) and 

Desulfuromonas group (Desulfuromonas sp. strain BB1 and 

Desulfuromonas chloroethenica). These primers were 

obtained from Löffler and represent an updated methodology 

from the nested primer set reported in Löffler and others 

(2000), which was determined to have a specificity problem. 

Positive controls analyzed for each group were 16S fragments that had been cloned into a plasmid by Löffler. DNA 

negative controls also were analyzed.

Biodegradation Data Analysis

Because ground-water flow directions in discharge wetlands are predominantly vertically upward, biodegradation 

processes and rates in the wetland sediments can be evaluated using flow velocities and concentrations of VOCs and 

redox constituents along a vertical flowpath. A review of 

available data for wetlands in the Cluster 13, Lauderick 

Creek area, however, showed that water-quality and hydrologic data generally were collected only at one depth in the 

wetland sediment at all piezometer or other porewater sampling sites located in the wetland (General Physics Corporation, July 1999). Only one site, PW-12, had screens placed 

at three depths in the wetland sediment (fig. 4). PW-12 consists of a bundle of three Teflon tubes (0.64-cm (centimeter) 

outside diameter), each with an approximately 2.5-cm-long 

screened interval vertical. VOC and chloride concentration 

data collected from site PW-12 in October 1998 were used 

to estimate field biodegradation rates of TeCA at Lauderick 

Creek, using the same method previously used at the 

West Branch Canal Creek area (Lorah and others, 2003). 

Measured TeCA concentrations were corrected for the 

effects of advection, dispersion, and dilution by normalization with the conservative tracer chloride, using the correction method outlined by Wiedemeier and others (1998). 

This tracer-corrected rate can be assumed to represent biodegradation rates if volatilization and sorption effects are 

assumed to be negligible. Because the shallowest screen at 

PW-12 is at a depth of 1 m, volatilization is likely to be minimal. Sorption also would have a negligible effect on the 

estimated field rates if the plume is at steady state (Lorah and 

others, 2003; Chapelle and Bradley, 1998). The first-order 

biodegradation rate was estimated by linear regression of the 

natural logarithm of the normalized TeCA concentration 

against traveltime along the flowpath. The traveltime was 

calculated using the linear ground-water flow velocity measured in the wetland sediments at West Branch Canal Creek 

(Lorah and others, 1997) because wetland flow velocities 

were not available for Lauderick Creek. Linear groundwater flow velocities from 0.6 to 0.9 m/yr (meters per year) 

were estimated using flow-net analyses, measured horizontal 

hydraulic conductivities, and estimated vertical hydraulic 

conductivities at West Branch Canal Creek (Lorah and 

others, 1997). The maximum of this estimated velocity 

range (0.9 m/yr), which was used to estimate field degradation rates for TeCA at West Branch Canal Creek (Lorah and 

others, 2003), was also used in this report to estimate a degradation rate at Lauderick Creek.

Assessment of Wetland Microbial 

Communities

The TRFLP analyses of the wetland sediments at

Lauderick Creek showed that the bacterial and methanogen 

communities are similar to those observed at the West 

Branch Canal Creek wetland area. The TRFLP profiles for 

the bacterial communities on three sampling dates are shown 

in the Appendix. Each peak on the TRFLP profiles corresponds to a specific bacterial group, characterized by the fact 

that the restriction enzyme used in the DNA sample preparation produced the same DNA fragment length (measured by 

base-pair (bp) size on the x axis of the TRFLP profile). To 

compare the bacterial communities at the Lauderick Creek 

and West Branch Canal Creek wetland areas, the occurrence 

and peak area of three of the bacterial groups identified as 

important in the biodegradation of TeCA at West Branch 

Canal Creek are shown for wetland sediment samples collected from the two areas—bacterial groups that had a peak 

bp size from 89 to 90 (89/90), 172, or from 198 to 200 (198/

200) (figs. 5a–b). The three bacterial groups were shown to 

be potentially important in TeCA degradation reactions by 

their increase in prominence during production of specific 

daughter compounds in wetland sediment microcosms 

amended with TeCA (Lorah and others, 2003). The 89/90 

peak potentially is involved in the hydrogenolysis of TeCA 

to 112TCA; and the 172 and 198/200 peaks potentially are 

involved in the dichloroelimination reactions of TeCA to 

12DCE and 112TCA to VC (Lorah and others, 2003). The 

three peaks generally were detected at all sites where wetland sediment samples were collected at West Branch (Lorah 

and others, 2003) and at Lauderick Creek. At three of the 

nine sites sampled at West Branch Canal Creek, all three 

peaks had approximately equal prominence in the bacterial 

community, with percent peak areas between 3 and 5 percent 

(fig. 5a). At four sites sampled at West Branch Canal Creek, 

the 198/200 peak was more prominent than the 89/90 and 

172 peaks, with percent peak areas as high as about 14 percent (fig. 5a). The three bacterial peaks also were present in 

the Lauderick Creek wetland sediment, although their relative prominence in the bacterial community differs from that 

in the West Branch Canal Creek sediments. At Lauderick 

Creek, the 89/90 and 172 peaks had percent peak areas 

between 3 and 6 in several of the samples; this range is similar to the range observed at West Branch Canal Creek. 

However, the 172 peak had percent peak areas as high as 10 

or 11 in several samples, whereas the 198/200 peak had percent peak areas less than 3 in all sediment samples. 

Although the 172 and 198/200 peaks are believed to be associated with the same dichloroelimination reactions (Lorah 

and others, 2003), the predominant bacterial group involved 

appears to differ between the West Branch Canal Creek and 

Lauderick Creek wetland areas.
Assessment of Wetland Microbial Communities 9

The bacterial communities at West Branch Canal Creek 

and Lauderick Creek also showed similar seasonal changes. 

In general, bacterial biomass was lower in the summer than 

in the winter at West Branch Canal Creek, even though 

microbial activity generally increases with warmer temperatures (Lorah and others, 2003). The same general biomass 

decrease was observed at Lauderick Creek, resulting in 

lower percent peak areas of important degradation-association bacteria in the summer than in the winter at many sampling locations (figs. 6a–b). For example, the bacterial peak 

at 89/90 bp had percent peak areas of less than 1 in four of 

the summer samples, but had percent peak areas as high as 

about 6 in samples collected from the same sites in the winter (fig. 6a). Although the reason for this decrease in importance of bacterial groups in the summer is unknown, it is 

possible that the growth of the wetland vegetation in the 

summer affects the microbial community (Lorah and others, 

2003). In microcosms constructed with wetland sediment 

from West Branch Canal Creek and incubated at the same 

temperature, lower biodegradation rates of TeCA and its 

daughter products were observed when sediment was collected in the summer than in the winter/early spring (Lorah 

and others, 2003). The lower biodegradation rates measured 

in the summer microcosms support the observation that the 

TRFLP analyses indicate that important bacterial peaks are 

lower in the summer than in the winter. 

The analyses using primers specific for the dehalorespiring bacteria in the Dehalococcoides (table 2) showed a 

similar seasonal trend to that seen for the general bacterial 

population observed from the TRFLP profiles (figs. 6a–b).
10 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD

Dehalococcoides species were detected at two sites in the 

winter and spring but were not detected at any site in the 

summer sampling period (table 2). Desulfuromonas, however, was detected at the same four sites in the spring and the 

summer, and only two samples showed positive detections in 

the winter (table 2). The detection level of this qualitative 

procedure to detect these two groups of dehalorespiring bacteria is unknown; a more quantitative method could assist in 

discerning seasonal trends. These dehalorespiring bacteria 

also have been detected in the wetland sediments at West 

Branch Canal Creek (Lorah and others, 2003), although their 

seasonal distribution currently is unknown.

For the methanogen community, the relative proportion 

of Methanosarcinaceae, a family of methanogens that 

includes all those capable of utilizing acetate as a substrate, 

was evaluated (table 3). The Methanosarcinaceae are 

believed to be important in degradation of VC to non-chlorinated compounds in the wetland sediment at West Branch 

Canal Creek (Lorah and others, 2003). The percentage of 

Methanosarcinaceae in the total methanogen community 

ranged from 4.2 to 31 and averaged 14, 14, and 16, respectively, in winter, spring, and summer. The percentage of 

Methanosarcinaceae showed little seasonal change in these 

wetland sediments. The percentage of Methanosarcinaceae 

in the Lauderick Creek sediments was similar to that 

observed at the West Branch Canal Creek wetland area, 

which averaged 12 percent (table 4). Thus, both the bacteria 

and methanogen communities at the Lauderick Creek area 

generally are similar to those at the West Branch Canal 

Creek area.
Assessment of Wetland Microbial Communities 11

 

 

Table 2. Detections of dehalorespiring bacteria in the Dehalococcoides and Desulfuromonas groups in 

wetland sediment grab samples collected in the Cluster 13, Lauderick Creek area, 2001–02

[Positive signs (+) indicate that the bacteria group was detected in the sediment sample, whereas negative signs (-) indicate that it was 

not detected.]

Sediment

site number

Dehalococcoides Desulfuromonas

12/18/2001 4/9/2002 6/18/2002 12/18/2001 4/9/2002 6/18/2002

SS1 - - - - - -

SS2 - - - - - -

SS3 - - - - + +

SS4 - - - - - -

SS5 + + - - + +

SS6A + + - - - -

SS6B - - - - - -

SS7 - - - + + +

SS8 - - - + + +

SS9 - - - - - -

SS10 - - - - - -

Table 3. Abundance of Methanosarcinaceae as a 

percentage of the total methanogen 

community in wetland sediment grab 

samples collected in the Cluster 13, 

Lauderick Creek area, 2001–02

[Dashes indicate data were not available]

Sediment

site number

Percentage of Methanosarcinaceae

12/18/2001 4/9/2002 6/18/2002

SS1 15 – –

SS2 5.2 12 –

SS3 22 16 20

SS4 16 10 10

SS5 6.1 14 12

SS6A 14 7.8 11

SS6B 10 16 20

SS7 15 18 14

SS8 4.2 4.5 14

SS9 17 8.8 9.1

SS10 25 31 31

Table 4. Abundance of Methanosarcinaceae as a 

percentage of the total methanogen 

community in wetland sediment grab 

samples collected in the West Branch 

Canal Creek area, March 1999

[From Lorah and others, 2003]

Site 

number

Percentage of 

Methanosarcinaceae

WB19 14

WB23 11

WB24 12

WB30 15

WB35 13

WB36 6.1
12 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD

Assessment of Biodegradation of Chlorinated 

Volatile Organic Compounds

The similar microbial communities in the wetland sediments at Lauderick Creek and West Branch Canal Creek 

indicate that similar biodegradation processes are occurring 

in both areas. The distribution of chlorinated VOCs with 

depth in the wetland porewater at site PW-12 shows that 

TeCA is degrading in the Lauderick Creek area and producing daughter compounds in proportions similar to those 

observed in the West Branch Canal Creek area (figs. 7a–b). 

TeCA concentrations decreased by a factor of 10 with 

decreasing depth along a 2-m vertical profile in the wetland 

sediments at site PW-12 (fig. 7a). The production of daughter compounds observed along this profile could account for 

the entire decrease in TeCA concentration. At both the 

Lauderick Creek and West Branch Canal Creek areas, 

12DCE and VC are the predominant, persistent daughter 

compounds produced in the wetland porewater (figs. 7a–b), 

which is consistent with observations from microcosm 

experiments (Lorah and others, 1997; Lorah and Olsen, 

1999a; Lorah and others, 2003). Relatively low concentrations of 112TCA also are observed at both sites. Microcosm 

experiments with West Branch Canal Creek sediments 
Summary and Conclusions 13

indicate that 112TCA is produced from TeCA degradation at 

the same or a higher molar concentration than 12DCE, but 

that the 112TCA then is degraded more rapidly than the 

12DCE (Lorah and Olsen, 1999a; Lorah and others, 2003). 

Field and laboratory data from the West Branch Canal Creek 

area show that 112TCA is degraded to VC and 12DCA, but 

12DCA analyses were not reported for the wetland porewater at Lauderick Creek (General Physics Corporation, July 

1999). The relatively low proportion of TCE present in the 

wetland porewater at Lauderick Creek (figs. 7a–b) is consistent with results of microcosm experiments with the West 

Branch Canal Creek wetland sediments, which indicate that 

production of TCE accounts for less than 10 percent of the 

TeCA degraded (Lorah and others, 2003).

TeCA concentrations near the base of the wetland sediments at Lauderick Creek are about a factor of 10 higher 

than those generally observed in the West Branch Canal 

Creek sediments, and not all the TeCA has been degraded 

(or attenuated by other processes) by the time the shallowest 

sampling depth is reached (fig. 7a). However, the shallowest wetland porewater samples in the Lauderick Creek area 

were collected from a depth of about 1 m (General Physics 

Corporation, July 1999), whereas passive diffusion samplers, 

called peepers, were used at West Branch Canal Creek to 

obtain detailed vertical profiles of concentrations between 

0 and 60 cm (Lorah and Olsen, 1999a, b). The peeper profiles at West Branch Canal Creek typically have shown that 

total VOC concentrations are near or below detection levels 

before land surface is reached (Lorah and others, 1997; 

Lorah and Olsen, 1999a, b). Because of the higher TeCA 

concentrations at Lauderick Creek, it is unclear whether similar complete attenuation would occur before land surface (or 

the surface of the creek bottom sediments) is reached.

Using the wetland porewater flow velocity of 0.9 m/yr 

that was calculated for the West Branch Canal Creek area, a 

TeCA biodegradation rate for the Lauderick Creek wetland 

sediment was calculated from the measured TeCA concentrations at site PW-12 (fig. 8). These calculations give a 

first-order degradation rate constant of 0.0034 per day, 

which corresponds to a half-life of about 200 days. For the 

West Branch Canal Creek area, field-derived TeCA degradation rates ranged from 0.0065 to 0.0085 per day at four sites, 

corresponding to a half-life of about 100 days (Lorah and 

others, 2003). The estimated degradation rates for both 

areas, therefore, are within the same order of magnitude. If 

flow velocity was determined for the Lauderick Creek wetland sediments, more accurate degradation rates could be 

calculated. A flow velocity of 1.8 m/yr for Lauderick Creek 

sediments, rather than the estimated 0.9 m/yr, would result in 

the same TeCA degradation rates as those calculated from 

field data for the West Branch Canal Creek.

Summary and Conclusions

The U.S. Geological Survey conducted a preliminary 

evaluation of the microbial communities and biodegradation 

processes in the wetland sediments at the Cluster 13, 

Lauderick Creek area, Aberdeen Proving Ground, Maryland, 

where ground water contaminated with chlorinated volatile 

organic compounds (VOCs) discharges from a sand aquifer 

to the wetland. Preliminary data from the Cluster 13, 

Lauderick Creek wetland area were compared to the more 

extensive data available from ongoing U.S. Geological 

Survey (USGS) studies in the West Branch Canal Creek wetland area, Aberdeen Proving Ground, to evaluate the technical feasibility of using natural attenuation or enhanced 

bioremediation methods for the ground-water plumes at the 

Lauderick Creek area. At both the Lauderick Creek and 

West Branch Canal Creek sites, 1,1,2,2-tetrachloroethane 

(TeCA) is the major VOC present in the aquifer.

Microbial communities in the Cluster 13 wetland area 

were compared to available USGS data from the West 

Branch wetland study area to evaluate the presence or 

absence of critical microbial species involved in TeCA degradation. The microbial community analyses showed that 
14 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD

similar bacterial and methanogen groups are present in the 

wetland sediments of the Cluster 13, Lauderick Creek area 

and the West Branch Canal Creek area. The bacterial and 

methanogen groups that have been identified as potentially 

important in the degradation reactions at West Branch Canal 

Creek are present in the Lauderick Creek wetland sediments 

and show similar seasonal patterns. These results indicate 

that similar TeCA biodegradation reactions would occur in 

sediments in both areas, and examination of the vertical distribution of TeCA and its anaerobic degradation products in 

the wetland porewater at one site (PW-12) supported this 

hypothesis. The TeCA daughter compounds produced and 

their relative proportions are similar in wetland porewater at 

Lauderick Creek and West Branch Canal Creek. Flow 

velocity in the wetland porewater at Lauderick Creek is 

unknown. However, if the flow velocity is assumed to be 

the same as at West Branch Canal Creek, a TeCA degradation rate constant of 0.0034 per day is calculated using the 

porewater concentrations at site PW-12. This rate constant 

corresponds to a half-life of about 200 days, whereas the 

field-calculated half-life of TeCA in the West Branch Creek 

wetland porewater is about 100 days.

The similar microbial communities and biodegradation 

processes and rates at Lauderick Creek and West Branch 

Canal Creek indicate that natural attenuation could be a 

remediation method for VOCs discharging to the wetland 

and creek-bottom sediments. Additional data are required 

to confirm the technical feasibility of natural attenuation, 

however. TeCA concentrations discharging to the 

Lauderick Creek wetlands are about a factor of 10 higher 

than those at West Branch Canal Creek, and substantial concentrations of TeCA, 1,2-dichloroethene, and vinyl chloride 

remained at the shallowest depth sampled (0.9 meters) at the 

one site for which contaminant distributions were available 

along a vertical flowpath (PW-12). Additional porewater 

data on VOC distributions in the upper 1 meter of the wetland sediments are needed to confirm attenuation of TeCA 

and its daughter compounds. Seasonal data on VOC distributions in the wetland porewater are critical because the 

West Branch Canal Creek study has shown that biodegradation rates are slower and VOC concentrations are higher in 

the wetland porewater in the summer than in the winter/early 

spring. To accurately determine degradation rates, nested 

piezometers in the wetland sediments are needed to obtain 

hydrologic measurements for calculation of flow velocities.

In addition to natural attenuation, enhanced bioremediation and constructed wetlands are possible remediation 

methods for the contaminants discharging in the Lauderick 

Creek area. As part of an enhanced bioremediation study in 

the West Branch Canal Creek area, a mixed culture capable 

of rapid biodegradation of TeCA and its anaerobic daughter 

compounds has been derived from the natural microbial 

community in the wetland sediments. The similarity of the 

natural microbial communities at Lauderick Creek and West 

Branch Canal Creek indicates that the mixed culture derived 

from West Branch Canal Creek wetland sediments could be 

applied to the Lauderick Creek area. Enhanced bioremediation techniques may be applicable in “hotspots” such as seep 

areas in wetland areas and Lauderick Creek. Wetlands are 

not present, however, immediately downgradient from some 

of the most highly contaminated areas in the aquifer near 

the south tributary of Lauderick Creek (southeast of sites 

WLC-84 and WLC-85). Construction of wetlands in this 

area could allow natural attenuation of the VOCs in wetland 

sediments before discharge to Lauderick Creek. These 

possible natural, enhanced, and constructed wetlands 

remediation methods could be used in conjunction with 

source removal remediation techniques. If complete source 

removal is infeasible, the natural and enhanced wetlands 

could act as a final treatment step for the remaining contaminants flowing from the source areas, and for those contaminants present outside the source treatment areas.
References Cited 15

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16 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD

Appendix—Bacterial Profiles
Appendix 17
18 Microbial Communities and Biodegradation of Chlorinated VOCs at Cluster 13, Lauderick Creek Area, APG, MD
Appendix 19