- Email: [email protected]
Laboratory studies identify a colloidal groundwater tracer: implications for bioremediation
FEMS Microbiology Letters 148 (1997) 131^135
Laboratory studies identify a colloidal groundwater tracer: implications for bioremediation Janet M. Strong-Gunderson *, Anthony V. Palumbo Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831-6038, USA
Received 3 December 1996 ; accepted 10 December 1996
Abstract
We have identified and tested a new colloidal tracer for use in hazardous waste site characterization and other environmental applications. The tracer is primarily composed of the dead ice nucleating active (INA) bacterium Pseudomonas syringae. Assay conditions are simple and based on the observation of the freezing behavior of 10 Wl solution volumes. Under specific assay conditions ( 5 to 7³C) these drops freeze only if the tracer is present. The results are available within 3 min of sample collection thus, providing near real-time results. The tracer detection limits are in the range of ng/l, it is stable over a pH range of 2^11, and maintains its high activity in the presence of a variety of contaminant compounds. In a simple column experiment this colloid tracer eluted just prior to NaCl with little or no tailing.
3
Keywords :
3
Sodium chloride; Groundwater £ow; Fracture £ow; Ice nucleating active bacterium ; Microsphere bead
1. Introduction
Ice nucleating active (INA) bacteria (e.g. monas
syringae,
P.
£uorescens,
Erwinia
Pseudo-
herbicola,
etc.) belong to a unique group of biological nucleators that catalyze ice nucleation at temperatures as high as 2³C [1]. Under speci¢c and controlled assay conditions chlorinated tap water will not freeze until approx. 15³C and groundwaters will not freeze until approx. 10³C. Thus, at high subzero temperatures ( 2 to 5³C) water will supercool and remain as a liquid. These INA bacteria synthesize large, outer membrane proteins that function as a template for ice crystal seeds called `ice nuclei' [2^4]. Therefore, if
3
3
3
3 3
* Corresponding author. Tel.: +1 (423) 576 0179; fax: +1 (423) 574 0765; e-mail: [email protected]
INA bacteria are present in a groundwater sample, ice crystallization will occur at these high subzero temperatures. This unique ice nucleating phenomena is the key to using the INA bacteria as an innovative environmental tracer. INA bacteria are very speci¢c for ice nucleation and there are few potential interferences from other nucleators. Compounds such as silver iodide (used in cloud seeding) are not active until temperatures fall below about 5³C [5]. Although there are some very unique classes of steroids [6] that can also nucleate water at high subzero temperatures, the only common nucleator that will function between 2 and 5³C is ice itself. The relationship between INA bacteria and their ability to reduce the cold tolerance of plants has been well characterized in numerous reports [7^9].
3
3
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 6 ) 0 0 5 3 9 - 3
FEMSLE 7428 15-5-97
3
132
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135
These INA bacteria have also been characterized with regard to insect cold tolerance [10^14]. A comprehensive review of INA bacteria and their application to date can be found in a new book by Lee et al. [15]. This is the ¢rst report that demonstrates the application of INA bacteria as an environmental tracer. As a tracer it can be used for the evaluation of stream £ow dynamics, groundwater and contaminant transport, drilling £uid tracer, microbial transport through the subsurface, etc. The laboratory experiments described here were in direct support of a ¢eld test (manuscript in preparation) where the INA bacteria were used in combination with other solutes and colloidal tracers to measure groundwater velocities in a fractured matrix at Oak Ridge National Laboratory. 2. Materials and methods
The ice nucleating active bacteria used in these laboratory experiments were a commercially available, concentrated, freeze-dried and killed preparation of P. syringae (Snomax Technologies, Rochester, NY). This product typically serves as a highly e¤cient source of heterogeneous ice nuclei used at numerous ski resorts to make snow. The INA tracer/product concentration and detection limits were calculated from standard curves. A 1000 ppm (w/v) solution of tracer was dissolved in sterile water and the ice nucleating activity of the solutions measured using the drop freezing assay [16]. Brie£y, this assay consisted of placing ten, 10 Wl solution drops on an aluminum weigh boat. The weigh boat £oated on the surface of a glycol cooled bath set at 37³C which corresponded to a drop temperature of approx. 35³C. The drops (typically 10 drops/dilution) were observed for 3 min and all frozen drops were counted and recorded as the number positive/total drops. These results were compared to the negative controls (tap water or upgradient groundwater). Serial dilutions were performed and ice nucleation activity measured to determine the detection limit. The reproducibility of the assay does not signi¢cantly vary with di¡erent lot numbers of the INA product. The short-term stability of the INA tracer was
measured in the presence of several common groundwater contaminants. The initial INA concentration was about 100 ppm in a contaminant solution containing 1 ppm of the following: toluene, naphthalene, xylenes, carbon tetrachloride, methylene chloride or trichloroethylene. This 1 ppm contaminant concentration was chosen based on other experiments on-going in our laboratory and on the characteristics of a potential ¢eld site where the INA tracer was to be ¢eld tested. The e¡ects of higher contaminant concentrations and mixed contaminants (including radioactive samples) are currently under investigation. Since the contaminants tested were volatile, a modi¢cation of the drop freezing assay [5] was performed with a 100 Wl test solution in an eppendorf microcentrifuge tube. The samples were capped and suspended in the low temperature bath (37³C) and observed 3 min for ice nucleation. Samples were run in triplicate and compared to the negative controls of water and water/contaminant mixture. An INA tracer concentration (10 ppm) was assayed for stability over a wide pH range. The pH was adjusted using a strong acid or base in increments of 0.5 pH units from an initial value of 7 down to 2.0 and from 7 up to 11.0. The drop freezing assay was used to qualitatively determine ice nucleating activity throughout the ranges (n = 11 pH intervals). The transport characteristics of the INA tracer were tested in a small soil column. Rich organic soil primarily composed of A-horizon material (Walker Branch Watershed, Oak Ridge National Laboratory, Oak Ridge, TN USA) was packed into a 30 ml column and £ushed with sterile water. The void volume was estimated to be approx. 5 ml. The column was initially £ushed with sterile water. A 100 Wl aliquot of the tracer solution (100 ppm INA in 85% NaCl) was dispensed onto the top of the column. The column was £ushed with approx. 60 ml distilled water and 1 ml fractions were collected. The NaCl concentration was measured using a Waters HPLC. Solution samples were ¢ltered with a 3 mm, 0.45 Wm Acrodisc 3 CR PTFE. A 20 Wl sample injection loop and an IC-Pak In Exclusion 7.8 mmU15 cm column were used for the NaCl analysis. Total conductivity was measured with a Waters Conductivity Detector, model 430. The ice
FEMSLE 7428 15-5-97
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135
nucleating activity was measured on the low temperature bath using the drop freezing assay. 3. Results and discussion
These laboratory experiments support the use of INA bacteria as a quick and inexpensive tracer for a variety of environmental conditions. This tracer ful¢lls a need for a product that could simulate not only groundwater and contaminant transport but also bacterial transport (i.e. colloid transport) which is critical to in situ bioremediation activities. The detection limit for the INA tracer using the drop freezing assay was determined to be 0.000001% or 0.01 ppm (Fig. 1). In theory, the INA tracer has a detection limit of 1 bacterium or nucleation site per sample volume. Thus, the detection limit should be even lower in the 100 Wl tube assay (versus the drop freezing assay) which was used in the contaminant stability assay. Once ice crystal growth has been initiated (i.e. nucleation has occurred), crystal propagation occurs throughout the sample. This low detection limit is important to large scale characterization studies where in situ dilution factors may be subject to gross underestimation or unknown. This low detection limit also permits the determination of breakthrough over a wider range of dilution than for many other tracers. In addition to being very sensitive the sample analysis is very rapid. Qualitative results can be obtained within 3 min of sample collection by measuring the freezing point, thus providing near real-time answers.
Fig. 1. Ice nucleating activity of a 0.1% solution (w/v) in water. Serial dilutions were performed and the number of frozen droplets recorded.
133
Table 1 Qualitative analysis of the ice nucleating activity (INA) stability in the presence of various contaminants Solution makeup Contaminant 1 ppm 100 Wl samples at 35³C Water unfrozen Water+INA frozen Water+INA toluene frozen Water+INA naphthalene frozen Water+INA xylenes frozen Water+INA carbon tetrachloride frozen Water+INA methylene chloride frozen Water+INA TCE frozen Water+contaminant tested individually unfrozen Initial tracer concentration at 1000 ppm.
Quick response times are important during dynamic ¢eld operations such as forced bacterial injection in bioremediation activities, pump and treat, etc. Results from the INA tracer can also be used to guide the sampling and analysis for other injected materials that may be costly and labor intensive. Site characterization activities that require repeated investigation will bene¢t from the use of this natural and biodegradable tracer. Changes in groundwater £ow velocities and £ow paths may differ from wet to dry seasons, or may be impacted by remediation activities such as pump and treat, etc. The ability to use the same tracer under a variety of conditions provides continuity in data interpretation. Common dye tracers (rhodamine, eosine, £uorosine etc.) are not readily biodegradable and residual concentrations may persist for several years, thus precluding their use more than once within a give region/site (W. Sanford et al., personal communication). Room temperature experiments have shown that within 4^8 weeks the INA tracer activity begins to decrease. This overall decrease in ice nucleating activity is dependent on temperature and natural microbial populations which can utilize the INA tracer (dead P. syringae) as a food source. Currently, there is a signi¢cant amount of labile carbon in the commercially available product that can be metabolized by the natural microbial populations. Thus, it is imperative that care must be taken to avoid well plugging and other problems if this commercial product is used in the ¢eld. Modi¢cations of the product to increase its suitability as a
FEMSLE 7428 15-5-97
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135
134
taminants do not interfere with the assay method. On-going experiments are evaluating contaminant mixtures as well as the long-term stability (months^ years) of the INA tracer under a variety of simulated ¢eld conditions and groundwater chemistries. The INA tracer was stable over the pH range 2.3^ 11.0. Thus, its application can be extended from pristine and organic contaminated sites to low pH sites such as acid mine drainage regions. Typically, the pH of these locations can range from 2 to 4 [17^ 19] with few tracers available which are stable under these extreme conditions and detectable at low concentrations. In an e¡ort to establish the transport characterFig. 2. Ice nucleating activity and NaCl breakthrough curves for
istics of this tracer a NaCl (solute) and INA (colloid)
the tracer experiments performed in a soil column.
mixture were £ushed through a soil column. Sequential one ml aliquots were assayed for both INA and NaCl. Analysis of the INA/NaCl data show an ini-
general environmental tracer are on-going in our lab-
tial breakthrough at 1 and 3 ml which is at the de-
oratory.
tection
limit
for
each
tracer
(Fig.
2).
De¢nitive
The optimal tracer for hazardous waste site char-
breakthrough occurred at 6 ml for the INA tracer
acterization activities needs to be easy, quick, eco-
and 11 ml for the NaCl tracer. The similar transport
nomical and cannot be a¡ected by the presence of
of the solute and colloid is common in a nonreactive
organic
compounds.
and homogeneous matrix and the e¡ect of matrix
High background or inherent levels of solutes and
di¡usion (at this scale) between the tracers and soil
contaminants
of
appears to be minimal. However, in a fractured sys-
many typical solute tracers such as dyes, bromide,
tem, the colloid tracer can elute ahead of a soluble
sodium chloride, etc. Under these conditions the ini-
tracer. Toran and Palumbo [20] reported that micro-
tial tracer loading/injection concentration could ne-
sphere beads transported ahead of the conservative
cessitate very high levels which could negatively im-
salt tracer due to preferential £ow paths and low
pact the dynamics of the system under investigation.
matrix di¡usion. After approx. 20^25, 1 ml aliquots
A ¢eld test has recently been completed at ORNL
the NaCl tracer returned to the baseline while the
and
inorganic
can
contaminant
impact
the
detection
limits
s
which examines the transport characteristics of the
INA tracer did not return to its baseline until
INA tracer compared to microsphere beads, live bac-
ml. This increased persistence of the INA tracer is
teria, bacteriophage, dyes, solutes and nobel gases.
due in part to the lower detection limits. There was
The preliminary data shows estimated groundwater
little to no additional tailing observed for the INA
velocities ranging from a few millimeters per day
tracer.
(solute based estimates) to several feet per day (colloid based estimates).
50
The laboratory work described here has identi¢ed the potential applications of these INA bacteria as
The addition of several contaminants at 1 ppm
an environmental colloidal tracer [21]. This work
(Table 1) did not interfere with the INA drop freez-
was in support of a recently completed ¢eld test at
ing assay when performed within 12 h. These con-
ORNL comparing the transport characterization of
taminants
chosen
these INA bacteria to other solute and colloidal trac-
based on the site at ORNL where the INA tracer
ers. The preliminary data indicate faster ground-
was scheduled as part of a site characterization.
water velocities when measured with the INA tracer
These experimental results support the potential use
than previously estimated using solute tracers that
of the INA tracer in hazardous waste site character-
interact and di¡use into the soil matrix. A more
izations. Under the conditions described here, con-
complete measure of the subsurface complexity can
and
their
concentrations
were
FEMSLE 7428 15-5-97
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135 be obtained by using multiple tracers : both solute
135
ness and Freezing Stress (Sakai, A. and Li, P.H., Eds.), Vol. 2, pp. 395^416, Academic Press, New York.
and colloid.
[9] Hirano, S.S., Nordheim, E.V., Arny, D.C. and Upper, C.D. (1982) Lognormal distribution of epiphytic bacterial populations on leaf surfaces. Appl. Environ. Microbiol. 44, 695^700.
Acknowledgments
[10] Strong-Gunderson, J.M., Lee, R.E. and Lee, M.R. (1989) Ice regulating bacteria promote transcuticular nucleation in in-
Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research, Corp., under
sects. Cryobiology 26, 551. [11] Strong-Gunderson, J.M., Lee, R.E., Lee, M.R. and Riga, T.J. (1990) Ingestion of ice nucleation active bacteria increases the
contract DE-AC05-96OR22464 with the US. Depart-
supercooling point of the lady beetle
ment of Energy. This research was sponsored by the
J. Insect Physiol. 36, 153^157.
In-Situ Remediation Technology Development Program of the O¤ce of Technology Development. Environmental Sciences Division publication number 4487.
[12] Strong-Gunderson, J.M., Lee, R.E. and Lee, M.R. (1992) Topical application of ice nucleating bacteria decreases insect cold tolerance. Appl. Environ. Microbiol. 58, 2711^2716. [13] Lee, M.R., Lee, R.E. and Strong-Gunderson, J.M. (1992) Isolation of ice nucleating active bacteria from a freeze-tolerant frog : Identi¢cation of
Pseudomonas putida
strains active in ice
nucleation. In : 29th Annual Meeting of the Society for Cryo-
References
biology, Ithaca, New York, June 14^19, 1992. Cryobiology 29, 759.
[1] Lindow, S.E., Arny, D.C., Barchet, W.R. and Upper, C.D. (1978) The role of bacterial ice nuclei in frost injury to sensitive plants. In : Plant Cold Hardiness and Freezing Stress (Li, P., Ed.), pp. 249^263, Academic Press, New York. [2] Warren, G., Corotto, L. and Wolber, P. (1986) Conserved repeats in diverged ice nucleation structural genes from two species of
Hippodamia convergens.
Pseudomonas.
Nucl. Acids Res. 14, 8047^8060.
[3] Warren, G.J. and Wolber, P.K. (1987) Heterogeneous ice nucleation by bacteria. Cryo Lett. 8, 204^215. [4] Mueller, G.M., Wolber, P.K. and Warren, G.J. (1990) Clustering of ice nucleation protein correlates with ice nucleation activity. Cryobiology. 27, 416^422. [5] Vali, G. (1995) Principles of ice nucleation. In : Biological Ice Nucleation and Its Applications, (Lee Jr., R.E., Warren, G.J. and Gusta, L.V., Eds.), pp. 1^28, APS Press, Minneapolis, MN. [6] Lee, R.E, Lee, M.R. and Strong-Gunderson, J.M. (1993) Review. Insect cold-hardiness and ice nucleating active microorganisms including their potential use for biological control. J. Insect Physiol. 39, 1^12. [7] Lindow, S.E., Lahue, E., Govindarajan, A.G., Panopoulos, N.J. and Gies, D. (1989) Localization of ice nucleation activity and the iceC gene product in Pseudomonas syringae and Escherichia coli. Mol. Plant-Microbe Interact. 2, 262^272. [8] Lindow, S.E. (1982) Population dynamics of epiphytic ice nucleation active bacteria on frost sensitive plants and frost con-
[14] Lee, R.E., Lee, M.R. and Strong-Gunderson, J.M. (1995) Biological control of insect pests using ice-nucleating microorganisms. In : Biological Ice Nucleation and Its Applications (Lee, R.E., Jr., Warren, G.J. and Gusta, L.V., Eds.), pp. 257^269, APS Press, Minneapolis, MN. [15] Lee, R.E., Warren, G.J. and Gusta, L.V. (1995) Biological Ice Nucleation and Its Applications, APS Press, Minneapolis, MN. [16] Vali, G. (1971) Quantitative evaluation of experimental results on
the
heterogeneous
freezing
nucleation
of
supercooled
liquids. J. Atmos. Sci. 28, 402^409. [17] Keswick, B.H. (1984) Sources of groundwater pollution. In : Groundwater Pollution Microbiology (Bitton, G. and Gerba, C.P., Eds.), pp. 39^64. Kriieger Publishing Co., Malabar, FL. [18] Nordstrom, D.K. (1982) Aqueous pyrite oxidation and the consequent formation of secondary iron minerals in acid sulfate weathering. SSSA Special Publication 10 (Acid Sulfate Weathering), pp. 37^62, Soil Science Society of America. [19] Barnes, H.L., Romberger, S.B. (1968) Chemical aspects of acid mine drainage. J. Water Pollut. Control Fed. 40, 371^ 384. [20] Toran,
L.
and
Palumbo,
A.V.
(1992)
Colloid
transport
through fractured and unfractured laboratory sand columns. J. Contaminant Hydrol. 9, 289^303. [21] Strong-Gunderson, J.M. and Palumbo, A.V. (1996) Biological tracer method. Patent Pending.
trol by means of antagonistic bacteria. In : Plant Cold Hardi-
FEMSLE 7428 15-5-97
Laboratory studies identify a colloidal groundwater tracer: implications for bioremediation Janet M. Strong-Gunderson *, Anthony V. Palumbo Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831-6038, USA
Received 3 December 1996 ; accepted 10 December 1996
Abstract
We have identified and tested a new colloidal tracer for use in hazardous waste site characterization and other environmental applications. The tracer is primarily composed of the dead ice nucleating active (INA) bacterium Pseudomonas syringae. Assay conditions are simple and based on the observation of the freezing behavior of 10 Wl solution volumes. Under specific assay conditions ( 5 to 7³C) these drops freeze only if the tracer is present. The results are available within 3 min of sample collection thus, providing near real-time results. The tracer detection limits are in the range of ng/l, it is stable over a pH range of 2^11, and maintains its high activity in the presence of a variety of contaminant compounds. In a simple column experiment this colloid tracer eluted just prior to NaCl with little or no tailing.
3
Keywords :
3
Sodium chloride; Groundwater £ow; Fracture £ow; Ice nucleating active bacterium ; Microsphere bead
1. Introduction
Ice nucleating active (INA) bacteria (e.g. monas
syringae,
P.
£uorescens,
Erwinia
Pseudo-
herbicola,
etc.) belong to a unique group of biological nucleators that catalyze ice nucleation at temperatures as high as 2³C [1]. Under speci¢c and controlled assay conditions chlorinated tap water will not freeze until approx. 15³C and groundwaters will not freeze until approx. 10³C. Thus, at high subzero temperatures ( 2 to 5³C) water will supercool and remain as a liquid. These INA bacteria synthesize large, outer membrane proteins that function as a template for ice crystal seeds called `ice nuclei' [2^4]. Therefore, if
3
3
3
3 3
* Corresponding author. Tel.: +1 (423) 576 0179; fax: +1 (423) 574 0765; e-mail: [email protected]
INA bacteria are present in a groundwater sample, ice crystallization will occur at these high subzero temperatures. This unique ice nucleating phenomena is the key to using the INA bacteria as an innovative environmental tracer. INA bacteria are very speci¢c for ice nucleation and there are few potential interferences from other nucleators. Compounds such as silver iodide (used in cloud seeding) are not active until temperatures fall below about 5³C [5]. Although there are some very unique classes of steroids [6] that can also nucleate water at high subzero temperatures, the only common nucleator that will function between 2 and 5³C is ice itself. The relationship between INA bacteria and their ability to reduce the cold tolerance of plants has been well characterized in numerous reports [7^9].
3
3
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 6 ) 0 0 5 3 9 - 3
FEMSLE 7428 15-5-97
3
132
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135
These INA bacteria have also been characterized with regard to insect cold tolerance [10^14]. A comprehensive review of INA bacteria and their application to date can be found in a new book by Lee et al. [15]. This is the ¢rst report that demonstrates the application of INA bacteria as an environmental tracer. As a tracer it can be used for the evaluation of stream £ow dynamics, groundwater and contaminant transport, drilling £uid tracer, microbial transport through the subsurface, etc. The laboratory experiments described here were in direct support of a ¢eld test (manuscript in preparation) where the INA bacteria were used in combination with other solutes and colloidal tracers to measure groundwater velocities in a fractured matrix at Oak Ridge National Laboratory. 2. Materials and methods
The ice nucleating active bacteria used in these laboratory experiments were a commercially available, concentrated, freeze-dried and killed preparation of P. syringae (Snomax Technologies, Rochester, NY). This product typically serves as a highly e¤cient source of heterogeneous ice nuclei used at numerous ski resorts to make snow. The INA tracer/product concentration and detection limits were calculated from standard curves. A 1000 ppm (w/v) solution of tracer was dissolved in sterile water and the ice nucleating activity of the solutions measured using the drop freezing assay [16]. Brie£y, this assay consisted of placing ten, 10 Wl solution drops on an aluminum weigh boat. The weigh boat £oated on the surface of a glycol cooled bath set at 37³C which corresponded to a drop temperature of approx. 35³C. The drops (typically 10 drops/dilution) were observed for 3 min and all frozen drops were counted and recorded as the number positive/total drops. These results were compared to the negative controls (tap water or upgradient groundwater). Serial dilutions were performed and ice nucleation activity measured to determine the detection limit. The reproducibility of the assay does not signi¢cantly vary with di¡erent lot numbers of the INA product. The short-term stability of the INA tracer was
measured in the presence of several common groundwater contaminants. The initial INA concentration was about 100 ppm in a contaminant solution containing 1 ppm of the following: toluene, naphthalene, xylenes, carbon tetrachloride, methylene chloride or trichloroethylene. This 1 ppm contaminant concentration was chosen based on other experiments on-going in our laboratory and on the characteristics of a potential ¢eld site where the INA tracer was to be ¢eld tested. The e¡ects of higher contaminant concentrations and mixed contaminants (including radioactive samples) are currently under investigation. Since the contaminants tested were volatile, a modi¢cation of the drop freezing assay [5] was performed with a 100 Wl test solution in an eppendorf microcentrifuge tube. The samples were capped and suspended in the low temperature bath (37³C) and observed 3 min for ice nucleation. Samples were run in triplicate and compared to the negative controls of water and water/contaminant mixture. An INA tracer concentration (10 ppm) was assayed for stability over a wide pH range. The pH was adjusted using a strong acid or base in increments of 0.5 pH units from an initial value of 7 down to 2.0 and from 7 up to 11.0. The drop freezing assay was used to qualitatively determine ice nucleating activity throughout the ranges (n = 11 pH intervals). The transport characteristics of the INA tracer were tested in a small soil column. Rich organic soil primarily composed of A-horizon material (Walker Branch Watershed, Oak Ridge National Laboratory, Oak Ridge, TN USA) was packed into a 30 ml column and £ushed with sterile water. The void volume was estimated to be approx. 5 ml. The column was initially £ushed with sterile water. A 100 Wl aliquot of the tracer solution (100 ppm INA in 85% NaCl) was dispensed onto the top of the column. The column was £ushed with approx. 60 ml distilled water and 1 ml fractions were collected. The NaCl concentration was measured using a Waters HPLC. Solution samples were ¢ltered with a 3 mm, 0.45 Wm Acrodisc 3 CR PTFE. A 20 Wl sample injection loop and an IC-Pak In Exclusion 7.8 mmU15 cm column were used for the NaCl analysis. Total conductivity was measured with a Waters Conductivity Detector, model 430. The ice
FEMSLE 7428 15-5-97
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135
nucleating activity was measured on the low temperature bath using the drop freezing assay. 3. Results and discussion
These laboratory experiments support the use of INA bacteria as a quick and inexpensive tracer for a variety of environmental conditions. This tracer ful¢lls a need for a product that could simulate not only groundwater and contaminant transport but also bacterial transport (i.e. colloid transport) which is critical to in situ bioremediation activities. The detection limit for the INA tracer using the drop freezing assay was determined to be 0.000001% or 0.01 ppm (Fig. 1). In theory, the INA tracer has a detection limit of 1 bacterium or nucleation site per sample volume. Thus, the detection limit should be even lower in the 100 Wl tube assay (versus the drop freezing assay) which was used in the contaminant stability assay. Once ice crystal growth has been initiated (i.e. nucleation has occurred), crystal propagation occurs throughout the sample. This low detection limit is important to large scale characterization studies where in situ dilution factors may be subject to gross underestimation or unknown. This low detection limit also permits the determination of breakthrough over a wider range of dilution than for many other tracers. In addition to being very sensitive the sample analysis is very rapid. Qualitative results can be obtained within 3 min of sample collection by measuring the freezing point, thus providing near real-time answers.
Fig. 1. Ice nucleating activity of a 0.1% solution (w/v) in water. Serial dilutions were performed and the number of frozen droplets recorded.
133
Table 1 Qualitative analysis of the ice nucleating activity (INA) stability in the presence of various contaminants Solution makeup Contaminant 1 ppm 100 Wl samples at 35³C Water unfrozen Water+INA frozen Water+INA toluene frozen Water+INA naphthalene frozen Water+INA xylenes frozen Water+INA carbon tetrachloride frozen Water+INA methylene chloride frozen Water+INA TCE frozen Water+contaminant tested individually unfrozen Initial tracer concentration at 1000 ppm.
Quick response times are important during dynamic ¢eld operations such as forced bacterial injection in bioremediation activities, pump and treat, etc. Results from the INA tracer can also be used to guide the sampling and analysis for other injected materials that may be costly and labor intensive. Site characterization activities that require repeated investigation will bene¢t from the use of this natural and biodegradable tracer. Changes in groundwater £ow velocities and £ow paths may differ from wet to dry seasons, or may be impacted by remediation activities such as pump and treat, etc. The ability to use the same tracer under a variety of conditions provides continuity in data interpretation. Common dye tracers (rhodamine, eosine, £uorosine etc.) are not readily biodegradable and residual concentrations may persist for several years, thus precluding their use more than once within a give region/site (W. Sanford et al., personal communication). Room temperature experiments have shown that within 4^8 weeks the INA tracer activity begins to decrease. This overall decrease in ice nucleating activity is dependent on temperature and natural microbial populations which can utilize the INA tracer (dead P. syringae) as a food source. Currently, there is a signi¢cant amount of labile carbon in the commercially available product that can be metabolized by the natural microbial populations. Thus, it is imperative that care must be taken to avoid well plugging and other problems if this commercial product is used in the ¢eld. Modi¢cations of the product to increase its suitability as a
FEMSLE 7428 15-5-97
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135
134
taminants do not interfere with the assay method. On-going experiments are evaluating contaminant mixtures as well as the long-term stability (months^ years) of the INA tracer under a variety of simulated ¢eld conditions and groundwater chemistries. The INA tracer was stable over the pH range 2.3^ 11.0. Thus, its application can be extended from pristine and organic contaminated sites to low pH sites such as acid mine drainage regions. Typically, the pH of these locations can range from 2 to 4 [17^ 19] with few tracers available which are stable under these extreme conditions and detectable at low concentrations. In an e¡ort to establish the transport characterFig. 2. Ice nucleating activity and NaCl breakthrough curves for
istics of this tracer a NaCl (solute) and INA (colloid)
the tracer experiments performed in a soil column.
mixture were £ushed through a soil column. Sequential one ml aliquots were assayed for both INA and NaCl. Analysis of the INA/NaCl data show an ini-
general environmental tracer are on-going in our lab-
tial breakthrough at 1 and 3 ml which is at the de-
oratory.
tection
limit
for
each
tracer
(Fig.
2).
De¢nitive
The optimal tracer for hazardous waste site char-
breakthrough occurred at 6 ml for the INA tracer
acterization activities needs to be easy, quick, eco-
and 11 ml for the NaCl tracer. The similar transport
nomical and cannot be a¡ected by the presence of
of the solute and colloid is common in a nonreactive
organic
compounds.
and homogeneous matrix and the e¡ect of matrix
High background or inherent levels of solutes and
di¡usion (at this scale) between the tracers and soil
contaminants
of
appears to be minimal. However, in a fractured sys-
many typical solute tracers such as dyes, bromide,
tem, the colloid tracer can elute ahead of a soluble
sodium chloride, etc. Under these conditions the ini-
tracer. Toran and Palumbo [20] reported that micro-
tial tracer loading/injection concentration could ne-
sphere beads transported ahead of the conservative
cessitate very high levels which could negatively im-
salt tracer due to preferential £ow paths and low
pact the dynamics of the system under investigation.
matrix di¡usion. After approx. 20^25, 1 ml aliquots
A ¢eld test has recently been completed at ORNL
the NaCl tracer returned to the baseline while the
and
inorganic
can
contaminant
impact
the
detection
limits
s
which examines the transport characteristics of the
INA tracer did not return to its baseline until
INA tracer compared to microsphere beads, live bac-
ml. This increased persistence of the INA tracer is
teria, bacteriophage, dyes, solutes and nobel gases.
due in part to the lower detection limits. There was
The preliminary data shows estimated groundwater
little to no additional tailing observed for the INA
velocities ranging from a few millimeters per day
tracer.
(solute based estimates) to several feet per day (colloid based estimates).
50
The laboratory work described here has identi¢ed the potential applications of these INA bacteria as
The addition of several contaminants at 1 ppm
an environmental colloidal tracer [21]. This work
(Table 1) did not interfere with the INA drop freez-
was in support of a recently completed ¢eld test at
ing assay when performed within 12 h. These con-
ORNL comparing the transport characterization of
taminants
chosen
these INA bacteria to other solute and colloidal trac-
based on the site at ORNL where the INA tracer
ers. The preliminary data indicate faster ground-
was scheduled as part of a site characterization.
water velocities when measured with the INA tracer
These experimental results support the potential use
than previously estimated using solute tracers that
of the INA tracer in hazardous waste site character-
interact and di¡use into the soil matrix. A more
izations. Under the conditions described here, con-
complete measure of the subsurface complexity can
and
their
concentrations
were
FEMSLE 7428 15-5-97
J.M. Strong-Gunderson, A.V. Palumbo / FEMS Microbiology Letters 148 (1997) 131^135 be obtained by using multiple tracers : both solute
135
ness and Freezing Stress (Sakai, A. and Li, P.H., Eds.), Vol. 2, pp. 395^416, Academic Press, New York.
and colloid.
[9] Hirano, S.S., Nordheim, E.V., Arny, D.C. and Upper, C.D. (1982) Lognormal distribution of epiphytic bacterial populations on leaf surfaces. Appl. Environ. Microbiol. 44, 695^700.
Acknowledgments
[10] Strong-Gunderson, J.M., Lee, R.E. and Lee, M.R. (1989) Ice regulating bacteria promote transcuticular nucleation in in-
Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research, Corp., under
sects. Cryobiology 26, 551. [11] Strong-Gunderson, J.M., Lee, R.E., Lee, M.R. and Riga, T.J. (1990) Ingestion of ice nucleation active bacteria increases the
contract DE-AC05-96OR22464 with the US. Depart-
supercooling point of the lady beetle
ment of Energy. This research was sponsored by the
J. Insect Physiol. 36, 153^157.
In-Situ Remediation Technology Development Program of the O¤ce of Technology Development. Environmental Sciences Division publication number 4487.
[12] Strong-Gunderson, J.M., Lee, R.E. and Lee, M.R. (1992) Topical application of ice nucleating bacteria decreases insect cold tolerance. Appl. Environ. Microbiol. 58, 2711^2716. [13] Lee, M.R., Lee, R.E. and Strong-Gunderson, J.M. (1992) Isolation of ice nucleating active bacteria from a freeze-tolerant frog : Identi¢cation of
Pseudomonas putida
strains active in ice
nucleation. In : 29th Annual Meeting of the Society for Cryo-
References
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