EPA/600/A-92/024
3rd Annual Symposium
GULF COAST HAZARDOUS SUBSTANCE
RESEARCH CENTER
BIOREMEDIATION
Fundamentals & Effective
Applications
PROCEEDINGS
February 21-22, 1991

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tttUS
MICROBIAL TRANSPORT THROUGH POROUS MEDIA: THE EFFECTS OF
HYDRAULIC CONDUCTIVITY AND INJECTION VELOCITY
H. J. MARLOW, K. L. DUSTON, M. R, WIESNER, M. B. TOMSON, /. T. WILSON AND C. H.
WARD
National Center for Ground Water Research
Department of Environmental Science and Engineering
Rice University
Houston, Texas
ABSTRACT
The effects of hydraulic conductivity and injection velocity on microbial transport through porous media
were investigated. Glass chromatography columns were packed separately with clean quartz sand of
two diameters (0.368 mm or 0.240 mm) and two hydraulic conductivities (1.37 x 10-1 cm/sec and 3.65
x 10-2 cm/sec respectively). Three injection velocities, 1.18 x 10-3, 2.35 x 10-3 and 4.73 x 10-3
cm/sec were investigated. Microbial transport under the conditions tested was limited and could be
predicted mathematically using a model for physicochemical filtration.
INTRODUCTION
The fate and transport of microorganisms in the subsurface environment has been the subject of research
for decades (1, 2, 3, 4). Early efforts were mainly concerned with public health and the contamination
of drinking water supplies by pathogenic microorganisms (5,6,7,8). Later research was aimed at
industrial applications such as inoculation of microorganisms to achieve microbial enhanced oil recovery
(9) and hazardous waste site remediation (10). This work was motivated in part by interest in the
injection of microorganisms with novel metabolic capabilities to remediate hazardous waste sites as well
as by the importance of assessing the transport of pathogenic microorganisms to infiltration galleries
and wells used for drinking water supplies.
Seed microorganisms have been used for decades in the initiation of industrial fermentation and
wastewater treatment operations. The success of inoculation depends on several criteria: organisms
must retain their specialized metabolic capability; organisms must come into contact with the
contaminants and nutrients; and environmental conditions must be conducive to contaminant
biodegradation and microbial survival (11). These criteria can be controlled in bioreactors, but may
be difficult, if not impossible, to control in expansive natural ecosystems.
Considerable attention and publicity have been given to the potential use of microorganisms to combat
oil slicks in marine and freshwater environments. The technology of seeding selected bacteria and fungi
to oil spills was patented by Azarowicz and Bioteknika International Inc. (12). However, Gutnick and
Rosenberg (13) and Atlas and Bartha (14) found inoculation to be ineffective in reducing oil
contamination in marine environments. More optimism was expressed for seeding and nutrient
supplementation in more contained environments (12, 13,15).
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Inoculation of microorganisms into the subsurface for enhanced biorestoration is an emerging technology
that may have met with some success (10, 16, 17). In the majority of cases, the role of the introduced
organisms in degradation of contaminants cannot be determined because appropriate control plots were
not incorporated into the experimental designs and results were not quantitatively measured throughout
the course of the projects (18). A detailed laboratory investigation performed under conditions
approximating those in situ is needed to assess the kinetics of degradation, the potential for toxicity, the
nutritional requirements of the organisms in the subsurface (19), and the factors affecting transport and
attachment of organisms.
Microbial Transport and Attachment
Studies have shown a wide range of microbial mobility in the saturated subsurface and through porous
media (20, 21, 22, 23). It is difficult to elucidate the factors that affect this transport because of the
wide variety of experimental conditions, incompleteness of reports, and lack of appropriate controls in
the published research. Jang et al. (24) suggested that the rate of bacterial spreading in a formation is
affected by the following factors: the porous structure, mineral composition and the wettability of rock
minerals; the hydrodynamics of the aquifer; surface physicochemical and microbiological properties of
the system; and secondary effects, such as the straining and plugging of the smaller pores and the
aggregation of bacterial cells. The transport of microorganisms through porous media may be predicted
with mathematical models derived from physicochemical filtration theory (25).
Microorganisms can adhere to almost any surface in any environment, which affects both the
distribution and activity of the organisms (26, 27). Microbial transport is dependent on the retention
of the microorganisms through attachment to the surfaces of the media particles. However, the
complexity and heterogeneity of aqueous and soil environments have made it extremely difficult to study
events related to the attachment of microorganisms in the subsurface. ZoBell (28) first suggested that
sorption occurred in two phases, an early, reversible sorption where bacteria are weakly held to the
surface and a later irreversible sorption that involved a more permanent attachment of the organism to
the surface. It is not known whether the primary factors controlling the degree of bacterial association
with surfaces are related to the capacity of the solid interfaces to support growth, purely to physical-
chemical interactions, or to some combination of these factors. It is known that microbial attachment
may be influenced by the nutrient concentration, ionic strength, production of biopolymers, presence
of surface tension depressants or detergents, effects of velocity and shear forces, integrity of the cell
wall, temperature (29), extracellular polysaccharides (30), and cellular appendages (31). Martin (32)
summarized a variety of investigations where electrostatic and hydrophobic forces emerged as potential
determinants of initial, reversible microbial attachment while irreversible attachment was controlled by
the formation of extracellular polysaccharides.
Models for Particle Deposition In Porous Media
Models based on a physicochemical description of particle deposition in porous media have been applied
to water filtration and may also be useful in estimating the retention of microbes in subsurface
environments. Particle deposition may be considered as a two-step process of transport and attachment.
Important transport mechanisms for particles in the size range of 10-2 to 102^m include interception,
gravity, and Brownian diffusion. A particle following a streamline within the porous medium that
passes within a distance of one particle radius from the sand grain will be intercepted by the aquifer
material. Alternatively, suspended particles, such as microbial organisms, may exit the fluid streamline
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and come in contact with sand grains as the result of Brownian or gravitational forces. In addition,
London-van der Waals and electrical double layer forces are likely to be important at small separation
distances between the particle and the medium and must be considered in modeling the particle
attachment step. In the absence of all forces other than interception, contact between suspended
particles and the porous medium may still occur.
The collector efficiency of a gran of filter medium or aquifer material is defined as the rate at which
particles strike the grain (collector) divided by the rate at which particles approach the collector. The
collector efficiency of a filter grain can be modified to include a parameter, a, that describes the fraction
of collisions between particles and filter grains resulting in particle attachment, yielding the single
collector removal efficiency, hr. The collision efficiency factor, a, is considered to be a function of
surface interactions between the particle and the collector and, therefore, is affected by the solution
chemistry.
The single-collector removal efficiency, hr, can be included in a mass balance on particles in an
incremental slice of the porous medium and integrated to yield an expression for the fraction of particles
remaining in suspension after passing a distance, L, through the porous medium:
n/no=exp [a3L(l-f)hr/2dc] (1)
where n and no are the effluent and influent concentrations of particles respectively,/is the porosity
of the medium, and dc is the average diameter of the medium. Equation 1 may be useful in estimating
the fraction of organisms remaining in suspension after vertical passage through aquifer media of
homogeneous composition that is well described by an average diameter. Under these conditions, the
potential for success in the inoculation of an aquifer for the purposes of bioremediation might be
assessed. The effects of hydraulic conductivity on microbial transport are contained in the estimates
of aquifer porosity and media size. The roles of injection velocity, microbial size, and density in the
transport and retention of microorganisms are captured in the calculation of hr, which is derived from
a consideration of particle transport by Brownian diffusion, gravity and interception.
EXPERIMENTAL SYSTEMS DESIGN
This research was designed to quantify the effects of injection velocity and hydraulic conductivity on
the movement of microorganisms through the subsurface. Rhodotorula sp., a species of yeast found
in ground water (33), was selected from a number of candidate organisms due to its shape and surface
characteristics. A bench-scale physical model was used to collect data, which were evaluated using a
model of physicochemical filtration. A similar approach has been used by others (25, 32, 34) in an
attempt to describe the distance bacteria can travel in an idealized, one-dimensional porous medium.
Details of the experimental design and variables for each of the experiments are shown in Table 1.
Hie average linear velocities (volumetric flux/porosity x cross sectional area, or Q/nA) investigated
ranged from 3.00 x 10-3 to 1.20 x 10-2 cm/sec, a factor of four. These injection velocity values
corresponded to ground water velocities that range from 2.59 m/day to 10.36 m/day (8.55 ft/day to
34.21 ft/day), values reported for both natural ground water flow and forced gradient tracer studies (4,
22, 23). The calculated hydraulic conductivity values were 1.37 x 10-1 and 5.59 x 10-2 cm/s
respectively for 45 and 70 mesh sand. The lower value of hydraulic conductivity, 5.59 x 10-2 cm/sec,
corresponded to a value reported for the hydraulic conductivity at the site of an aviation gasoline spill
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in Traverse City, Michigan (35). The higher value of hydraulic conductivity, 1.37 x 10-lcm/sec,
corresponded to a value reported for the hydraulic conductivity at a site in Cape Cod, Massachusetts.
This site, described by Harvey et al. (4), is the focus of an active research effort to describe the
movement of microorganisms through the saturated subsurface.
The experimental apparatus consisted of a high capacity infusion/ withdrawal syringe pump (Harvard
Apparatus, South Natick, MA) connected with 1/16 in. i.d. Teflon tubing to duplicate chromatography
columns (Spectrum® Medical Industries, Inc., Los Angeles, CA) filled with sand. The columns were
connected to a fraction collector (Isco, Inc., Lincoln, NB) to collect the effluent. A simple water
manometer was connected to the inlet and outlet of each column to determine the pressure differential
during the experiments (Figure 1).
RESULTS AND DISCUSSION
Tritium Breakthrough Curves
The combined tritium breakthrough curves for all experiments are presented in Figure 2. The tritium
concentration measured in the effluents from the duplicate columns (identified as L and R for left and
right) were expressed as fractions of the influent concentrations in each experiment and were plotted
as a function of the number of pore volumes eluted from the columns. Inspection of the plot revealed
a sigmoidal breakthrough curve with a midpoint (n/no=0.5) equal to one pore volume, which is
characteristic of uniform plug flow through homogenous packed porous media. The similarity in the
curves indicated the reproducibility of flow characteristics and column packing between experiments.
Effect of Injection Velocity
The results from experiments examining the effect of flow rate on the transport of Rhodotorula sp. are
summarized in Figure 3. Data obtained from duplicate columns were not statistically different (at a
95% confidence interval). At a given filtration rate, microorganisms began to break through the
columns after approximately 0.5 pore volumes were eluted, roughly corresponding to the breakthrough
curves produced using tritiated water. At all three injection velocities, microbial numbers in the
effluents continued to increase slowly after the passage of one pore volume and appeared to level off
after 8 to 10 pore volumes. An increase in the injection velocity resulted in a decrease in the retention
of microorganisms by the porous media. For the 45 mesh sand (K = 1.37 x 10 -1 cm/s), a doubling of
the flow rate nearly doubled the number of organisms transported through the column. The observed
retention of Rhodotorula sp. as a function of flow rate corresponds well with the retention of
microorganisms calculated from equation 1.
Effect of Hydraulic Conductivity
The results from experiments examining the effect of hydraulic conductivity on the transport of
Rhodotorula sp. are summarized in Figure 4. At given hydraulic conductivity values, microbial
breakthrough after approximately 0.5 pore volumes roughly corresponded to the breakthrough curves
produced using tritiated water. The microbial numbers in the effluents continued to increase slowly and
appeared to level off after 8 to 10 pore volumes. A reduction of the hydraulic conductivity by about
a third resulted in a tenfold reduction in the number of organisms transported. Again, experimental
trends are predicted accurately by the physicochcrnical model for particle deposition (equation 1).
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CONCLUSIONS
The results of this research indicate that inoculation of an aquifer with microorganisms could be best
performed by creating conditions of high injection velocity in a formation of great hydraulic
conductivity. However, the most significant increases in the transport of microbial cells are likely to
be brought about through manipulation of the collision efficiency, a. It may be possible to alter the
surface properties of the organism by, among other things, controlling the ionic strength of the solution,
by varying the nutritional status of the organism, and/or by introducing "stabilizing agents" to the
injection mixture (32).
Under conditions likely to exist in most ground waters, retention of microorganisms should be high, and
their transport should be limited in the absence of inhomogeneities in the aquifer material, such as
cracks and channels. The majority of an aquifer may not be perfused by an injection of microorganisms
when transport is controlled by flow through preferential paths. Therefore, the ability to successfully
seed and bioremediate an aquifer may be limited by the "filtration" of microorganisms into relatively
homogeneous portions of the aquifer. The Rhodotorula sp. was predicted to penetrate porous media
(45-1.6) approximately 80 cm with a decrease in microbial numbers of five orders of magnitude.
Growth of the microorganism, which is likely to enhance microbial transport over longer periods of
time, is not taken into account by this model. Harvey et al. (4) and Martin (32) have speculated on the
importance of growth and shedding or sloughing of cells for the colonization of porous media by
microorganisms.
DISCLAIMER
Although the research described in this article was supported by EPA through Assistance Agreement
No. CR-812808 to Rice University, it has not been subjected to Agency review and therefore does not
necessarily reflect the views of the Agency, and no official endorsement should be inferred.
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TECHNICAL REPORT DATA
{Please read Instructions on the reverse before cample''
1. REPORT NO. 2,
EPA/600/A-92/024 *
3,
4. title ano subtitle
MICROBIAL TRANSPORT THROUGH POROUS MEDIA: THE EFFECTS
OF HYDRAULIC CONDUCTIVITY AND INJECTION VELOCITY
S. REPORT DATE
6. PERFORMING ORGANIZATION COOE
7. AUTHORIS) .
H.J. MARLCWr M.R. WIESNER1 ' C.H. WARD1
K.L. DUSTON1 M.B. TOMSON1 J.T. WILSON2
8. PERFORMING ORGANIZATION REPORT NO.
9_J>ERFORMING ORGANIZATION NAME AND AOORESS
1RICE UNIVERSITY, HOUSTON, TX 77005
%.S. EPA, RSKERL, ADA, OK 74820
10. PROGRAM ELEMENT NO.
CBWD1A
11. CONTRACT/GRANT NO.
DW14934013
12. SPONSORING AGENCY NAME ANO AOORESS
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY - ADA
U.S. ENVIRONMENTAL PROTECTION AGENCY
P.O. BOX 1198
AM, OK 74820
13. TYPE OF REPORT ANO PERIOD COVERED
SYMPOSIUM PAPER
14. SPONSORING AGENCY CODE
EPA/600/15
15. SUPPLEMENTARY NOTES
PUBLISHED: BIOREMEDIATION: FUNDAMENTALS & EFFECTIVE APPLICATIONS, PROCEEDINGS 3RD
ANNUAL SYMPOSIUM GULF COAST HAZARDOUS SUBSTANCE RESEARCH CENTER, PP 75-82, FEB. 21-22, 199:
16. ABSTRACT
THE EFFECTS OF HYDRAULIC CONDUCTIVITY AND INJECTION VELOCITY ON MICROBIAL TRANSPORT
THROUGH POROUS MEDIA WERE INVESTIGATED. GLASS CHROMATOGRAPHY COLUMNS WERE PACKED
SEPARATELY WITH CLEAN QUARTZ SAND OF TOO DIAMETERS (0.368 MM OR 0.240 MM) AND TWO
HYDRAULIC CONDUCTIVITIES (1.37 x 10-1 CM/SEC AND 3.65 x 10-2 CM/SEC RESPECTIVELY).
THREE INJECTION VELOCITIES, 1.18 x 10-3, 2.35 x 10-3 AND 4.73 x 10-3 CM/SEC WERE
INVESTIGATED. MICROBIAL TRANSPORT UNDER THE CONDITIONS TESTED WAS LIMITED AND COULD
BE PREDICTED MATHEMATICALLY USING A MODEL FOfc PHYSICOCHEf'ICAL FILTRATION.
17.	KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATi Field,Group
MICROORGANISMS
TRANSPORT
POROUS MATERIALS
POROUS MEDIA
FILTRATION
MICROBIAL-TRANSPORT
MATHEMATICAL-MODEL

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