Miscible Displacement through Gamma Radiation-Sterilized Soil Columns J. C. Corey,l D. R. Nielson,2 J . C. Picken, Jr., and Don Kirkham Iowa State University, Ames, Iowa RUBBER SEPTUMS
Gamma radiation from a 6OCo source has proved successful in sterilizing soil columns. Sterile miscible displacement methods were used to follow the displacement of nitrate at slow velocities through soils. After a soil column was sterilized, 100 % of the nitrate added to a Webster soil-sand column was recovered in the effluent after displacement at 0.055 cm. per hour, while 66 of the nitrate was recovered when displaced through a nonsterile column at the same velocity.
TO FRACTION-
COLLECTOR
cm
RESERVOIR
M
iscible displacement has been used to study the movement of inorganic ions through soil inasmuch as the method permits one to study the influence of soil, water content, and flow velocity of water on ion movement. In previous studies (Day, 1956; Handy, 1959; Kaufman and Orlob, 1956; Nielson et al., 1964; Sadler et al., 1965) the microbial population of the soil was of little consequence in determining the final recovery of the inorganic salts. With the current emphasis on research into the movement of biodegradable soil additives such as pesticides, the contribution of microbial activity to the ultimate fate and location of these soil additives must be determined. Because of the heterogeneous and dynamic character of soil microbial populations, a critical examination of the degradation of an additive moving in soil-water requires that the population be identified and its activity known. A convenient method of controlling the microbial population would be the inoculation of sterilized soil with desired microbial species. This paper presents a method whereby soil can be not only sterilized and inoculated but can be used in a procedure to conduct long term, controlled flow experiments with soil additives that are decomposed or altered by microbial activity. In the authors’ method, gamma radiation from a 6oCo source is used to sterilize the soil and the miscible displacement methods of Nielsen and Biggar (1961) are used to control the flow velocity of the solutions. When the microbial population is eliminated by means of the ionizing radiation, an opportunity is available to study the interaction of inorganic or organic substances in the soil without fear of net changes in concentration owing to the activity of microorganisms. Although nitrate is used as an illustrative ion in this paper of a substance whose movement is influenced by microbial activity, the method is applicable to any biodegradable substance that can be prepared in a sterile form. Experbnentul Procedure
Acrylic plastic cylinders (Figure 1) 7.5 cm. in diameter and 12.5 cm. in length were sealed with marine resin to input end plates consisting of an acrylic plastic housing, a fritted glassbead plate, and a reservoir whose openings were sealed with Present address, Radiological and Environmental Sciences Division, E. I. du Pont de Nemours & Co., Aiken, S. C. 2 Present address, Department of Water Science and Engineering, University of California, Davis, Calif. 144 Environmental Science and Technology
cm FOR REMOVAL OF AIR BUBBLES
Figure 1. Acrylic plastic column used in miscible displacement experiments
rubber septums. The size of the column was restricted by the chamber within the radiation field of an available 8500-curie 6oCosource of the Iowa State University Medical Research Institute. Prior to irradiation, the plastic cylinders were packed uniformly with a soil-sand mixture. This soil-sand mixture comprised equal weights of a washed sand (1.00 to 0.5 mm. in size) and a Webster clay loam surface soil (37.5 % sand, 26.4% silt, 36.6% clay, pH 5.9, 2.0% organic carbon, and 0.16% nitrogen) that had been passed through a 2-mm. sieve. After the soil-sand mixture within the cylinder had been water-saturated with a dilute solution of 0.005M Cas04 to prevent deflocculation, the effluent end plate was sealed in position, the column sterilized if required, and a miscible displacement experiment conducted. The soil column was sterilized by exposure to gamma radiation from the ‘ T o source for 3.5 hours. The dose distribution within the column varied depending upon the location within the column (Figure 2). The dose distribution was obtained using glass dosimeters spaced throughout the water-saturated soil column (Davidson er al., 1956). This distribution within the system had to be known so that the column could be irradiated long enough to ensure a sterilizing dose in it everywhere. A dose in excess of 1,000,000 rads (rad is the dose of any ionizing radiation which produces the energy absorption of 100 ergsper gram) is required for sterility (Eno and Popenoe, 1964). A miscible displacement experiment consisted of displacing 50 ml. of distilled water containing 0.275 gram of CaCl2 and 0.175 gram of Ca(NO&. 4 H 2 0 through the water-saturated soil at a velocity of 0,055 cm. per hour with 0.005M CaSO4. The effluent samples were analyzed for nitrate using the steamdistillation method of Bremner and Keeney (1965) and for chloride using Mohr’s silver nitrate method. Breakthrough
-
STERILE SOLUT'oY
-
1
STERILE COTTON
20,000
Figure 3. Schematic drawing of apparatus used in the sterile and nonsterile miscible displacement experiments 18,000
Table I. Physical Data for the Miscible Displacement Experiments on Sterile and Nonsterile Webster Soil (A Horizon) and Silica Sand (These data correspond to Figures 4, 5, and 6 ) Figure 5 Figure 6 Figure 4 Webster A Webster A Webster A Sanda Sanda Soil Sanda
14,000
Figure 2. Dose distribution (in rads/ minute) within the acrylic plastic column when the column was irradiated in the 6oCo facility
curves, plots of relative concentrations (C;C,) (where C is the concentration of chloride or nitrate found in the effluent and COis the concentration of chloride or nitrate added to the column) against the volume of effluent collected, were made from these data. Figure 3 illustrates the apparatus used in the miscible displacement experiments. Sterile plastic tubing (Administration Set No. 8200, Sigmamotor, Middleport, N. Y . )was used to conduct water solutions to the column. The tubing had a screw-type adapter at one end to connect it to a sterilized supply bottle containing sterile solution and an adapter at the other end to connect it to a sterile hypodermic needle. A constant flow pump (Sigmamotor, No. TM-20-2) was used to regulate the flow of solution through the tube. Solution was removed from the column by means of a hypodermic needle inserted through the rubber septum located at the center of the effluent end plate. The effluent was collected automatically with a fraction collector. No attempt was made to flush the areas behind the plate on the influent side during changes of solution required in the procedure since this operation would enhance the possibility of contamination. F o r sterile work o r for work involving selected microbial species, sterile solutions and sterile tubing should be used to prevent the introduction of undesired species. Results The nitrate-chloride solution was displaced through soil columns in three separate experiments to show the effectiveness of gamma-radiation and aseptic methods for obtaining and maintaining sterile conditions within the soil column. In the first experiment, 50 ml. of the chloride-nitrate solution was displaced through a column of nonsterile soil (Table I) in order to determine the amount of nitrate lost under nonsterile conditions. The area under the breakthrough curves (Figure 4) from this experiment represents the amounts of chloride and nitrate recovered in the eWuent. If no chloride
+
+
Bulk density, g./cm. Bulk volume, cm. Pore space volume, ~ m Porosity, cm. 3/cm. Volume of H,O in sample, ml. Column length, cm. Column cross-sectional area, cm. Column position Flow rate of effluent, ml. /hr . Velocity of flow, cm./hr. Sterile
Nitrate lost, 0
zof added
+
1.478 575 197 . ~ 0.343
1.508 575 192 0.334
1.478 575 197 0.343
197 12.5
192 12 5
197 12.5
45.6 Vertical
45.6 Vertical
45.6 Vertical
2.5
2.5
2.5
0,055 No
0.055 Yes
0
34
The Webster soil and silica sand
iii
0,055 Originally (but inoculated) 37
equal weight percentage.
or nitrate is lost while passing through the column, the recovery should represent the amount of chloride or nitrate initially added to the column in the 50-ml. volume. In this experiment, the entire amount of chloride was recovered but only 6 6 z of the nitrate. The breakthrough curve for nitrate is approximately symmetrical and within the chloride breakthrough curve. The symmetrical nature of the nitrate curve implies that the microbial population remained relatively constant throughout the experiment. In a second experiment an irradiated soil column was used with aseptic techniques to illustrate the efllectiveness of the suggzsted method for maintaining sterile conditions. The breakthrough curve for nitrate (Figurz 5) is similar to the chloride curve but is displaced slightly to the right, indicating a later arrival in the effluent. No losses of nitrate or chloride occurred during displacement. At the completion of the experiment, the column was dismantled and soil from various locations within the column was plated on egg albumin agar (Fred and Waksman, 1928). N o bacterial or actinomycete Volume 1, Number 2, February 1967 145
q o.6
1 '
I
'5
0
WEBSTER t SAND NON-STERILE
2
-0
I-
2I-
E
0.4
1 WEBSTERt SAND INOCULATED
1
0.4
2 W
0
z
0
0 W
0.2
2
3a W
0
20 0 300 0 VOLUME (ml) Figure 4. Breakthrough curves for a 50-ml. aqueous solution of chloride and nitrate displaced at 0.055 cm./hr. through a 12.5-cm. long column of Webster soil and silica sand (50/50 by weight) 100
6 -6.0 I \
i
I
I I
WEBSTER t SAND STERILE
?!Z
I
"0-
IO 0
200
300
VOLUME (ml) Figure 5. Breakthrough curves for a 50-ml.aqueous solution of chloride and nitrate displaced at 0.055 cm./hr. through a sterilized soil column
colonies appeared in or on the agar after incubation for 7 days at 24" C. The third experiment was designed to determine the feasibility of inoculating a sterile soil column to establish a particular microbial population. The column used in the first experiment was irradiated for 3.5 hours and all procedures followed, except the hypodermic needle used to introduce solution to the column was first dipped into a nonsterile soil suspension before insertion into the column. A sterile 0.005M CaS04 solution was passed through the inoculated column for 12 hours at a flow velocity of 1.32 cm. per hour before introducing 50 ml. of the solution containing nitrate and chloride. The nitrate and chloride solution was introduced at a velocity of 0.055 cm. per hour (Table I). The breakthrough curves for this experiment (Figure 6) showed a loss of 37z of the added nitrate during the passage of nitrate through the column, but no loss of chloride. I n comparison to the curves in Figure 4 (nonsterile condition), the nitrate curve in Figure 6 is translated to the left in relation to that of chloride. The nonsymmetrical nature of the nitrate curve implies that the microbial population was increasing rapidly throughout the experiment. 146 Environmental Science and Technology
VOLUME (ml) Figure 6. Breakthrough curves for a 50-ml. aqueous solution of chloride and nitrate displaced at 0.055 cm.,ihr. through an inoculated soil column
Preliminary results from previous experiments have shown that the shape of the breakthrough curves for chloride was not influenced when the soil column was exposed to radiation for periods between 0 and 5 hours. A comparison of the chloride breakthrough curves in Figures 4 and 6 illustrates this observation for periods of 0 and 3.5 hours.
Discussion Denitrification of added nitrate normally prevents the study of nitrate movement at small flow velocities through saturated surface soils. To overcome this experimental problem chloride has often been used to represent the movement of nitrate (Stephens, 1962; Wetselaar, 1961). As the study of the movement through, and interaction with, soil of more complicated molecules is attempted, it will be difficult, if not impossible, to find a material that will represent the behavior of the biodegradable compound in the soil but that at the same time will not be influenced by microbial activity itself, To obviate this problem, an experimenter using the method outlined is able to conduct an experiment to determine the influence of microbial activity as well as soil on the movement of the soil additive and then sterilize the column and repeat the experiment to determine the influence of soil alone o n its movement. This experimental procedure avoids the difficult problems involved in preparation of duplicate columns for soil-water movement studies. Radiation methods are ideally suited for this type of experiment since sterilization can be achieved without dismantling the apparatus. Although all living soil organisms were killed using the sterile column technique, the possibility of enzyme systems remaining active after irradiation was not tested. If enzymes are not completely inactivated, their action on biocides and other organic materials offers an additional consideration when using the technique. The sterile column technique should have a wide application in the study of the movement of oxygen, carbon dioxide, herbicides, fungicides, and waste products through soils. Experimental conditions would have to be more refined than those illustrated, when unsaturated flow or gas diffusion is occurring because the soil column would be exposed to the atmosphere during the experiment. Sterility could be easily maintained, however, by conducting the experiments within sterile chambers.
Literature Cited Bremner, J. M., Keeney, D . R., Anal. Chin?. Acta 32, 485-95 (1 965). Davidson, S., Goldblith, S. A., Proctor, B. E., Nucleonics 14, 34-9 (1956). Day, Paul R., Trans. Am. Geophys. Union 37, 595-601 (1956). Eno, Charles F., Popenoe, Hugh, SoilSci. Soc. Am. Proc. 28, 533-5 (1964). Fred, E. B., Waksman, S. A., “Laboratory Manual of General Microbiology,” McGraw-Hill, New York (1928). Handy, L. L., J. Petrol. Technol. 11 (3), 61-3 (1959). Kaufman, W. J., Orlob, G. T., Trans. Am. Geophys. Union 37, 297-306 (1956). Nielsen, D. R., Biggar, J. W., Soil Sci. SOC.Am. Proc. 25, 1-5 (1961).
Nielsen, D. R., Biggar, J. W., Miller, R. J., Calif. Agr. 18, 4-5 (1964). Sadler, Lloyd D. M., Taylor, S. A., Willardson, L. S., Keller, J., Soil Sci. 100, 348-55 (1965). Stephens, D., J . Soil Sci. 13, 52-9 (1962). Wetselaar, R., Plant Soil 15, 121-33 (1961). Receioed for reciew December 16, 1966. Accepted February I , 1967. Journal Paper No. J-5433 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No. 998. Contribution from the Department of Agronomy and Veterinary Medicine Research Institute, Iowa State Unicersity. Work supported by the U S . Atomic Energy Commission, Contract No. AT-(ll-I)-1269, Report No. COO-1269-14. The second author acknowledges support of a Senior PostDoctoral National Science Foundation Fellowship.
An Electron Microscope Study of Colloids in Waste Water R. B. Dean
US. Department of the Interior, Federal Water Pollution Control Administration, Cincinnati, Ohio 45226 Stig Claesson, Nils Gellerstedt, and Nils Boman Institute of Physical Chemistry, University of Uppsala, Uppsala, Sweden ~~
~
Electron micrographs of the colloidal fraction in the effluent from an activated sludge waste water-treatment plant show fragments of bacterial cell walls as the dominant material. Viruses, phage, flagella, and other cellular debris are present to a lesser degree. The cell wall fragments appear to have a thickness near 100 A. and a width from 500 to 5000 A. (0.05 to 0.5 micron). The fragments are loosely clumped together in preparations which have been freeze-dried at -75 o C. but are flattened out and almost invisible in preparations dried near room temperature. Clarification by membrane filtration, or by flocculation with lime or ferric chloride, removes most of the colloidal material of cellular origin.
T
he colloidal fraction of waste waters from sewage treatment plants has not previously been observed directly. Bishop et al. (1965, 1967) have demonstrated that colloidal organic matter will not be removed on columns of activated carbon in reasonable periods of time. Organic material associated with previous use of waste water must be removed before the water can be judged fit for intentional re-use. The only materials of colloidal dimensions previously identified in treated waste water are viruses and humic substances (Clarke and Kabler, 1964; Wedgwood, 1952). Rudolfs and Balmat (1952) made electron micrographs of the colloids in raw sewage but do not appear to have used freeze-drying. It has been assumed that cellular debris will be present, but its quantity and structure have been strictly matters for conjecture. After this work was completed, Rickert and Hunter (1966) reported their work on high speed centrifugation of sewage effluents. The authors have made electron microscope observations of the colloidal constituents of waste water from a modern activated sludge treatment plant using low temperature freeze-drying to preserve the structure (Claesson et al., 1967). Solid matter in the size range from 0.01 to 1 micron was found to fall into two distinct groups: bacteria, viruses,
phages, and related compact bodies having a definite size and shape; and shapeless fragmentary material loosely clumped together. The principal component of this fragmentary group appears to be cell walls from bacteria, although other cellular debris is undoubtedly present. The aggregation of cell wall material is certainly produced in part by the operations necessary to prepare the specimens. However, the loose open nature of the fragmentary material demonstrates that the particles are not strongly flocculated in the starting material.
Technique
Secondary effluent was obtained from the Uppsala, Sweden, sewage treatment plant which was completed in 1957 and is currently being doubled in capacity. This is an activated sludge plant using step aeration. Operating difficulties have been minimal despite overloading and the presence of branchedchain alkylbenzene sulfonates (ABS) in the detergents. Effluent BOD averages 30 mg. per liter, corresponding to a removal efficiency of 88 %. In this work, effluent was immediately filtered through glass fiber filter mat 1106 BH (Mine Safety Appliances, Pittsburgh, Pa.) to remove suspended matter. A separate sample of this filtered product was sealed under vacuum and heated in boiling water for 1 hour before shipment to Cincinnati for chemical analysis (Table I). The total organic carbon (TOC) of 11 mg. per liter on the filtered material corresponds to about 30 mg. per liter as COD and about 20 mg. per liter as BOD (Williams, 1966). Uppsala has very hard water, ranging from 15 to 20 German degrees, corresponding to 5.3 to 7.1 meq. per liter with an alkalinity of 4.5 to 5.0 meq. per liter. The hardness of the effluent is correspondingly high. Total dissolved solids (TDS) may be estimated as about 490 mg. per liter based on the conductivity of 750 pmhos. Electron microscopic examination of organic colloids must be carried out on dry specimens. It is necessary to separate the colloids, which probably do not exceed 5 mg. per liter, from the large excess of dissolved salts which would otherwise obscure the field. Colloids were concentrated by centrifugation Volume 1. Number 2. February 1967 147
