101
REMEDIATION Autumn 2005
A new use for biofilm barriers was developed and successfully applied to treat nitrate-contami-
nated groundwater down to drinking water standards. The barrier was created by stimulating in-
digenous bacteria with injections of molasses as the carbon donor and a combination of yeast ex-
tract and trimetaphosphate as nutrients. This injection of amendments results in bacterial growth
in the aquifer, which attaches to the sand grains to create a reactive semipermeable biofilm. The
biofilm barrier presented in this article reduced the migration of contaminants and provided an
active zone for remediation. The cylindrical biobarrier was constructed using eight wells on the
perimeter forming a 60-foot-diameter reactive biodenitrification region. Another well at the cen-
ter was installed to continuously extract the treated water. The intent was to produce a continu-
ous source of nitrate-free water. The system operated for over one year, and during this period,
the biobarrier was revived multiple times by reinjecting molasses in the perimeter wells. Nitrate
concentrations of treated water decreased from 275 mg/L (as nitrogen) to 
 1 mg/L. © 2005 Wiley
Periodicals, Inc.
BACKGROUND 
Biofilm barriers are a promising new technology for the containment and remediation
of groundwater.These barriers are formed by subsurface injection of bacteria and/or
nutrient solutions to stimulate the growth of bacteria that produce extracellular poly-
meric substances (EPS) and also may degrade contaminants (Costerton et al., 1995).
The engineered accumulation of biomass and EPS in the subsurface is used to control
groundwater flow and enhance treatment strategies. For example, a biofilm barrier can
be used as a “funnel” to channel contaminated groundwater to a treatment zone. Unlike
many other barrier technologies, biofilm barriers cause minimal surface disturbance and
have no obvious depth limitation.
MSE Technology Applications (MSE), in collaboration with the Center for Biofilm
Engineering (CBE), has developed a biological process that combines containment and
treatment of contaminated groundwater.The process involves the creation of a subsur-
face biofilm barrier to destroy nitrate in situ while keeping the contaminated plume
from reaching fresh water sources. Containment is achieved by selectively plugging per-
meable strata with a microbial biofilm (Cunningham et al., 1997). As the biofilm devel-
ops, it reduces the subsurface hydraulic conductivity, attenuating the transport of con-
taminants. Several pilot-scale tests conducted by MSE and CBE at the demonstration site
in Butte, Montana (Exhibit 1), demonstrated that biobarriers act as an effective alterna-
tive to conventional barrier technologies. Both laboratory and pilot tests confirmed that
© 2005 Wiley Periodicals, Inc.
Published online in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/rem.20063
Lomesh Dutta
H. Eric Nuttall
Al Cunningham
Garth James
Randy Hiebert
In Situ Biofilm Barriers: Case Study 
of a Nitrate Groundwater Plume,
Albuquerque, New Mexico
although the biobarrier greatly reduced the hydraulic conductivity, it never completely
blocked the flow of water (Cunningham et al., 1991; James et al., 1995).
The current study is a collaborative project between MSE, CBE, and the University
of New Mexico (UNM).The study focuses on the denitrification of groundwater using
the biofilm biobarrier technology. Phase 1 of this project was conducted at a demonstra-
tion site in Butte, Montana (Cunningham et al., 2004) using an artificial sand lagoon. A
biobarrier was established across the center of the test cell that was 130 feet wide, 180
feet long, and 20 feet deep by injecting denitrifying bacteria as well as a molasses- and
nitrate-based nutrient solution.
The bacterial strain CPC211A of starved cell suspensions of Pseudomonas fluorescens
were injected to produce abundant amounts of extracellular mucoid exopolymers able to
grow well under simulated field conditions and metabolically aid in the denitrification re-
action (Hiebert et al., 2001; James et al., 1995, 2000). Microbial counts, including enu-
meration of heterotrophic bacteria and denitrifying bacteria, were performed using sam-
ples collected from the monitoring wells throughout the experiment from December
1999 to October 2001. Near the end of the experiment, counts of heterotrophic bacteria
were higher in samples from the centerline (C0–C10) wells than from the upstream
(T1–T3) and downstream (T4–T8) monitoring wells.The results indicate that the major-
ity of the biomass forming the biobarrier was located along the center of the test cell.
The barrier was maintained for over two years through the periodic injection of
small quantities of nutrient solution. Despite the high concentration of nitrate added to
the test cell, nitrate-nitrogen levels in the test cell effluent did not exceed the federal
maximum contaminant level (MCL) of 10 mg/L during the project (Exhibit 2).
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Exhibit 1. Phase 1 study test cell schematic showing monitoring well locations in Butte, Montana
FIELD DEMONSTRATION
Phase 2 of this project was conducted to demonstrate the field-scale application of per-
meable biofilm barriers for the denitrification of groundwater.
Objective
The objective was to develop a successful biobarrier at the field site and determine its
effectiveness in containment and remediation of nitrate in groundwater.The ability to
develop an effective biobarrier by stimulating the indigenous species was an important
consideration to determine the commercial applicability of the technology.
Site Description
The test site chosen for Phase 2 was the Mountain View Subdivision located in the South
Valley of Albuquerque, New Mexico.The nitrate contamination of groundwater was
caused by overfertilization of a vegetable farm in the 1950s.The nitrate plume covers an
area of about 550 acres and has a volume of approximately 6.4 billion liters (Deng,
1998; Nuttall, 2000).The nitrate concentrations at the site were measured to be on the
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© 2005 Wiley Periodicals, Inc. 103
Exhibit 2. Contour map of nitrate concentrations determined at monitoring wells located
throughout the test cell near the end of the experiment (September 24, 2001)
Note: The biofilm barrier (located ca. 60 feet west) was effective in removing nitrate from the groundwater.
order of 300 mg/L, which is 30 times more than the federal MCL of 10 mg/L. A
methemoglobinemia incident was reported at this site in the 1980s where an infant was
hospitalized in the Mountain View community due to consuming water from a private
well that contained high concentrations of nitrate.
Test Cell Preparation
A cylindrical zone of 3,000 square feet as shown in Exhibit 3 was designed.The zone was
based on the requirements to use both the containment capability and the reactivity of
the biofilm barrier to produce a system capable of efficiently remediating the nitrate-con-
taminated plume.The test pattern was composed of eight barrier construction wells (BC-
1–8) installed uniformly on the perimeter of the circular zone, nine monitoring wells set
up inside (CMW-1, CMW-2, and MW-E1), on (BMW-1 and BMW-2), and outside
(OMW-1, OMW-2, and MW-C1) the circular pattern, and a Center Well to continuously
produce clean water. An additional set of five wells (five spot [NE, NW, SE, SW, and
Center5]) located more than 300 feet from the circular zone, was used to supply nutri-
ent-free water to the cell.The five spot wells were previously used for denitrification ex-
periments at this field site (Chen, 2000; Deng, 1998; Nuttall et al., 1997).
Groundwater Flow Modeling 
Modeling was performed to determine the flow rates required to construct the in situ
biofilm barrier.Visual MODFLOWTM v.2.8.2 was used based on the site characteriza-
tion data. A 300-square-foot area was modeled in which the well system was designated
as the center of the system.The center zone containing the wells and the outer zone of
the barrier had cell sizes of 1 square foot and 10 square feet, respectively.The aquifer
zone was divided into two vertical layers.The upper one extended from the ground sur-
face to 32 feet below ground level (bgl), and the lower one ranged from 32 feet to 50
feet bgl.The conductivity of the upper layer was (Kupper): 1  10
–3 and the lower one
In Situ Biofilm Barriers
© 2005 Wiley Periodicals, Inc.104
Exhibit 3. The circular biobarrier design
was (Klower): 1  10
–2.The Specific Yield (Sy) and the Specific Storage (Ss) were 0.3 and
0.0001, respectively, and remained the same for both the layers.The groundwater gradi-
ent was 0.00001, resulting in h of 0.03 feet across the 300-foot modeled area.The nom-
inal groundwater level (GWL) was 34 feet bgl. For modeling, the GWL and gradient
constant head boundaries (CHBs) of 34.03 and 34.00 feet bgl were used at the left and
right edges of the modeled area.To provide initial wetting of the upper layer, an initial
head of 30 feet bgl was set for the modeled area and transient analysis was chosen
(Dutta, 2004).
Microbes and Nutrients
Groundwater samples were collected across the span of wells and were assayed for pop-
ulations of bacterial species present.The data were used to determine potential nutri-
ents required to stimulate indigenous bacterial species. Specifically, microbiological anal-
yses were performed at the site to evaluate heterotrophic plate counts (HPCs) (i.e.,
bacterial colonies per mL of sample); direct counts (DCs) (i.e., direct counting of each
cell under microscope); and denitrifying bacteria (DNB) most probable number (MPN)
estimates (i.e., statistical estimation of bacteria growing in the aqueous media). Bacteria
capable of utilizing nitrate and nitrite as terminal electron acceptors and also producing
copious amounts of extracellular polymers were desired for this project. Denitrifying
bacteria are known to be present ubiquitously, and thus producing a biofilm barrier
without the inoculum would result in considerable cost savings as well as prevent signifi-
cant regulatory hurdles. Previous tests at the site (Nuttall, 1997) had demonstrated in-
stances of occurrence of both denitrification and biofouling, which indicated that bacte-
ria capable of producing the desired results were already present.
Of the many possible carbon nutrients, including acetate, citrate, lactate, ethanol,
and molasses, the latter was selected based on the ability to stimulate production of an
effective biofilm as confirmed during the laboratory column experiments, previous ex-
perience of MSE in stimulating rapid bacterial growth with the production of mucoid
extracellular polymers using molasses solutions, and economic considerations.
Phosphate and yeast extract were mixed with molasses to produce the appropriate
nutrient solution to favor bacterial growth.The groundwater would supply the terminal
electron acceptor (nitrate) to complete the bacterial metabolism.
Biobarrier Formation
After installation and development of the wells, the nutrient solution was injected to
form the biobarrier. A total of 5 1/2 batches were prepared, with each batch having the
composition shown in Exhibit 4.
The barrier construction wells BC-1–8 and Center Well were used to create the
biobarrier in the aquifer.The injection process was performed June 4–June 10, 2003. A
schematic of the injection process is shown in Exhibit 5.
Initially, the barrier construction wells BC-2, BC-4, BC-6, and BC-8 were injected
with the nutrient mixture in 1:40 parts of molasses to water using a peristaltic pump set
to obtain an approximate molasses concentration of 4.0 g/L.The feed tank was used to
store the prepared batches of nutrient mixture, and the surge tank supplied clean water
to the injection operation from a location on the site as shown in Exhibit 5. A peristaltic
REMEDIATION Autumn 2005
© 2005 Wiley Periodicals, Inc. 105
Bacteria capable of utiliz-
ing nitrate and nitrite as
terminal electron accep-
tors and also producing
copious amounts of extra-
cellular polymers were
desired for this project.
pump was set at the rate of 0.6 gallons per minute (gpm), and the total feed rate in-
jected into each of the wells was maintained at approximately 5.5 gpm. During this
step, the barrier construction wells BC-1, BC- 3, BC-5, and BC-7 and the Center5 spot
well were constantly pumped and fed to the surge tank to maintain the mass balance of
water in the tank (flow in  flow out), thereby maintaining the tank volume.
After injecting the desired amount of nutrients into the wells BC-2, BC-4, BC-6, and
BC-8, the extraction pumps were pulled out and nutrients were then injected into wells
BC-1, BC-3, BC-5, and BC-7. In this case, the surge tank was filled using only the water
from the five spot wells. Hence, lower flow rates for injection had to be used.The injec-
tion process was followed by injecting “clean” water from the five spot wells into all BC
wells at ~4.5 gpm for 30 minutes to drive the nutrients from the inside and surroundings
of the well to prevent biofouling.Water at 290 mg/L from the five spot wells and free
from nutrients was continuously injected into the Center Well during all injection opera-
tions at a rate equivalent to the injection rate into the other wells.The process prevented
the nutrients from coming toward the Center Well, resulting in a biobarrier with the de-
sired circular pattern. At the end of the injection phase, the system was given a two-week
incubation period to allow the bacteria to establish the biobarrier.
In Situ Biofilm Barriers
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Exhibit 4. Concentrations of batch solutions during nutrient injection
Quantity
Ingredient Per batch Total
Molasses 47 gallons 258.5 gallons
Trisodium phosphate 13 lbs. 71.5 lbs.
Yeast extract 2–3 lbs. 11–16.5 lbs.
Water Filled up to a total Total of 2,200 lbs.
of 400 gallons
Exhibit 5. Flow diagram of nutrient injection in the field
Operating and Monitoring System
The Center Well was continuously used to produce treated water at 1 gpm. The
treated water was returned to the aquifer using injection well DW-1, located more
than 300 feet away from the region of the barrier as shown in Exhibit 6. This setup
resulted in a hydraulic gradient between the biobarrier zone and the surrounding
aquifer that allowed the continuous inflow of contaminated water across the denitri-
fying biobarrier.
Monitoring wells were installed inside (CMW-1, CMW-2, and MW-E1), on (BMW-
1 and BMW-2), and outside (OMW-1, OMW-2, and MW-C1) the circular biobarrier, as
shown in Exhibit 6. Groundwater samples from the Center Well and the monitoring
wells were assayed using YSI 6920 groundwater environmental monitoring system and
Dionex ion chromatography equipment over a period of several months for nitrate (as
nitrogen) concentrations.
RESULTS AND DISCUSSION
The nitrate values in the Center Well dropped from 275.1 mg/L nitrate (as nitrogen) at
the start of the experiment to a value of < 0.05 mg/L (MCL  10mg/L) during the
course of the operation.The concentrations of nitrate detected in samples from the
Center Well are shown in Exhibit 7.
As shown in Exhibit 7, the initial nitrate reduction was rapid. Nitrate-nitrogen val-
ues dropped from 275 mg/L to 64mg/L in the first 57 days.The subsequent rise may be
due to an organic carbon limitation in the biofilm barrier. Nutrients were reinjected
after intervals of 86 and 196 days from the start of the operation, also shown in Exhibit
7.The experiment had a total runtime of 401 days, 74 days of which the system re-
mained shut down.The cease in operations was due to two nutrient reinjection opera-
tions and biofouling in the Center Well, resulting in buildup of biomass on the Center
Well’s pump, which caused it to stop functioning.
The nitrate (as nitrogen) concentrations in the monitoring wells with time are
shown in Exhibit 8.The concentrations of nitrate detected in monitoring wells inside
(CMW-1, CMW-2, and MW-E1) the biofilm barrier dropped significantly, while the
REMEDIATION Autumn 2005
© 2005 Wiley Periodicals, Inc. 107
Exhibit 6. Schematic diagram showing location of wells
concentrations in the wells outside the barrier (OMW-1, OMW-2, and MW-C1) re-
mained consistent with the initial values.Thus, these results demonstrate the biofilm
barrier’s ability to denitrify groundwater.
OPERATIONAL EXPERIENCE/ISSUES
Biofouling
During the process, biofouling was seen as one of the major challenges to the operation.
The accumulation of biomass occurred around well screens as well as in the pipeline
In Situ Biofilm Barriers
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Exhibit 7. Nitrate (as nitrogen) concentration in the Center Well with time
Exhibit 8. Nitrate (as nitrogen) concentration in monitoring wells BMW-1, BMW-2, CMW-1,
CMW-2, MW-E1, OMW-1, OMW-2, and MW-C1
from the Center Well. Coating and clogging of surfaces resulted in an overflow of the
disposal well and temporary suspension of the operation of the pump used to extract
water from the Center Well.These fouling problems began occurring around four
months after the start of operation. Excess biomass was removed from the disposal well
and associated piping by using air pressure to lift the fouled water column out of the
well, then blowing the biomass out of the well. After blowing as much biomass as possi-
ble, the well was surged several times with water and bleach solution to destroy biomass
around the well bore. Biweekly additions of 1.5 gallons of bleach to both the disposal
and the Center Well were scheduled thereafter.
Nutrient Reinjection
To maintain the barrier, it had to be revived multiple times by adding the amendment
mixture into the eight perimeter wells. The amount of amendment injected and the
duration before each reinjection was calculated based on stoichiometric calculations.
The reinjection procedure was similar to the biobarrier formation process, except
that the continuous injection of nutrient-free water into the Center Well was not
performed.
SUMMARY AND CONCLUSIONS
An in situ biobarrier was developed at a site located in the South Valley of
Albuquerque, New Mexico.The curtain was developed by stimulating the indigenous
bacteria using molasses as the carbon substrate.The results of the study indicate that
the biobarrier was able to contain and remediate nitrate concentrations 30 times over
the MCL, thus demonstrating the technology as a promising approach for the treat-
ment of contaminated groundwater.The ability to direct groundwater flow using a
biobarrier could be used to channel contaminated groundwater to an active treatment
zone while also contributing to bioremediation of the water. In situations where
groundwater flow is minimal, pumping strategies to draw the contaminated ground-
water into an active treatment zone could be enhanced with biobarrier technology.
This technology has commercial value for assisting agricultural businesses, such as
feedlots, hog farms, and fertilizer suppliers in reducing their environmental impact
and ensuring the availability of safe drinking water. Since the barrier is constructed of
a very low-cost carbon source (i.e., molasses at $0.17/pound) and given that the bar-
rier can be rejuvenated many times, this technological approach to in situ bioremedia-
tion will have significant cost advantages.
REFERENCES
Chen, J. (2000). Continuous biodenitrification of groundwater: In situ field test. Unpublished master’s the-
sis, Chemical and Nuclear Engineering Department, University of New Mexico.
Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., & Lappin-Scott, H. M. (1995). Microbial
biofilms. Annual Review of Microbiology, 49, 711–745. 
Cunningham, A., Sharp, R., Hiebert, R., & James, R. (2004). Fundamentals and applications of starved cell
sub-surface biological barriers. Journal of Bioremediation. 7(4-3), 1–13. 
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Cunningham, A., Warwood, B., Sturman, P., Horrigan, K., James, G., Coesterton, J. W., et al. (1997). Biofilm
processes in porous media—Practical applications. In P. S. Amy & D. L. Haldesman (Eds.), The microbi-
ology of the terrestrial deep subsurface (pp. 325–344). Boca Raton, FL: Lewis Publishers.
Cunningham, A. B., Characklis, H. M., Abedeen, F., & Crawford, D. (1991). Influence of biofilm accumula-
tion on porous media hydrodynamics. Environmental Science and Technology, 25, 1305–1311.
Deng, L. (1998). In situ biological denitrification of groundwater. Unpublished master’s thesis, Chemical and
Nuclear Engineering Department, University of New Mexico.
Dutta, L. (2004). Pilot scale testing of in situ biofilm barrier for remediation of groundwater. Unpublished
master’s thesis, Chemical and Nuclear Engineering Department, University of New Mexico.
Hiebert, R., James, G., Sharp, R., & Cunningham, A. (2001, August). Development and Demonstration of
Subsurface Biofilm Barriers Using Starved Bacterial Cultures. Association of Environmental Health and
Sciences (AEHS) Contaminated Soil Sediment and Water, International Issue, pp. 45–50.
James, G. A., Warwood, B. K., Cunningham, A. B., Sturman, P. J., Hiebert, R., & Costerton, J. W. (1995).
Evaluation of subsurface biobarrier formation and persistence. Proceedings of the 10th Annual
Conference of Hazardous Waste Research, Great Plains–Rocky Mountain Hazardous Substance Research
Center, Manhattan, KS, pp. 82–91.
James, G. A., Warwood, B. K., Hiebert, R., & Cunningham, A.B. (2000). Microbial barriers to the spread of
pollution. In J. J. Valdes, Bioremediation (pp. 1–14). Boston: Kluwer Academic Publishers. 
James, G. A., Warwood, B. K, Horrigan, K., Hiebert, R., Cunningham, A. E., & Coesterton, J. W. (1996).
Containment of heavy metal and chlorinated organic solvent contamination using microbial barriers
formed in packed-sand columns. Presented at the 96th General Meeting of the American Society of
Microbiology, New Orleans, LA. Washington, DC: American Society for Microbiology.
Nuttall, H. E. (1997). Biodenitrification of New Mexico ground water. New Mexico Journal of Science, 3, 64–73.
Nuttall, H. E. (2000, June). Research and development. In Interstate Technology and Regulatory Council
(ITRC), Emerging technologies for enhanced in situ biodenitrification (EISBD) of nitrate-contaminated
groundwater: Technology overview (pp. 17–23). Washington, DC: ITRC. 
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water at an UMTRA site. International Symposium In Situ and On Site Bioreclamation Proceedings, pp.
435–440. 
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Lomesh Dutta works at Kleinfelder Inc. in Sacramento, California. Prior to this, he obtained a master’s
degree in chemical engineering from the University of New Mexico, where he worked with Dr. Eric Nuttall
on biofilm barriers as a part of his master’s degree research.
H. Eric Nuttall is a professor in chemical engineering at the University of New Mexico. His areas of re-
search include bioremediation and nanotechnology applications to water cleanup. He is active in the
Interstate and Technology Regulatory Council and teaches Internet training. Dr. Nuttall consults in the area
of in situ bioremediation.
Al Cunningham is a professor of civil engineering and also directs the Research and Education
Initiatives program for Montana State University's Center for Biofilm Engineering. He has developed a cross-
disciplinary research program in hydrodynamics and microbial process engineering with focus areas that in-
clude: bioremediation of contaminated groundwater and soil, bioavailability of organic compounds and met-
als, microbial transport, and biofilm accumulation in porous media and closed conduits. Current research
sponsors include the National Science Foundation, the Environmental Protection Agency, the Department of
Energy, the Army Research Office, MSE Technology Applications Inc., Conoco Inc., and the Inland Northwest
Research Alliance.  Dr. Cunningham has developed graduate- and undergraduate-level projects and courses
that investigate environmental microbial process problems of concern to industry and society. Industrial and
government participants provide suitable projects and progress reviews.
Garth James was a staff microbiologist at MSE Technology Applications Inc. in Bozeman, Montana, during
this project. Dr. James holds a BS degree in microbiology from the University of Saskatchewan and a PhD in
microbial ecology from the University of Calgary. Dr. James has extensive experience in environmental micro-
biology, biofilm microbiology, project management, and working with multidisciplinary teams. Dr. James is
currently employed by the Center for Innovation in Bozeman, Montana, and Montana State University.
Randy Hiebert, P.E., is the biotechnology manager at MSE Technology Applications Inc. He has both BS
and MS degrees in chemical engineering from Montana State University. Mr. Hiebert has been a project
manager for MSE on several projects, including the Mine Waste Technology Program Nitrate Destruction
Project. This project consisted of biologically destroying nitrate-contaminated wastewater emanating from
the TVX Mineral Hill Mine near Jardine, Montana. Mr. Hiebert was also the project manager for the
Department of Energy Biofilm Barrier Projects and several pollution prevention and waste minimization pro-
jects for the U.S. Army Construction Engineering Research Laboratory.
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