APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1993, p. 467-4720099-2240/93/020467-06$02.00/0Copyright © 1993, American Society for Microbiology

Protozoa in Subsurface Sediments from Sites Contaminatedwith Aviation Gasoline or Jet Fuel

JAMES L. SINCLAIR,`* DON H. KAMPBELL,2 MIKE L. COOK,2 AND JOHN T. WILSON2

ManTech Environmental Technology, Inc., Robert S. Kerr Environmental Research Laboratory, P.O. Box1198, Ada, Oklahoma 74820,1 and Robert S. Kerr Environmental Research Laboratory,

U.S. Environmental Protection Agency, P.O. Box 1198, Ada, Oklahoma 748202

Received 26 May 1992/Accepted 12 November 1992

Numbers of protozoa in the subsurface of aviation gasoline and jet fuel spill areas at a Coast Guard base atTraverse City, Mich., were determined. Boreholes were drilled in an uncontaminated location, in contami-nated but untreated parts of the fuel plumes, and in the aviation gasoline source area undergoing H202biotreatment. Samples were taken from the unsaturated zone to depths slightly below the floating free productin the saturated zone. Protozoa were found to occur in elevated numbers in the unsaturated zone, where fuelvapors mixed with atmospheric oxygen, and below the layer of floating fuel, where uncontaminatedgroundwater came into contact with fuel. The same trends were noted in the biotreatment area, except thatnumbers of protozoa were higher. Numbers of protozoa in some contaminated areas equalled or exceeded thosefound in surface soil. The abundance of protozoa in the biotreatment area was high enough that it would beexpected to significantly reduce the bacterial community that was degrading the fuel. Little reduction inhydraulic conductivity was observed, and no bacterial fouling of the aquifer was observed during biotreatment.

Protozoa have been found in the subsurface at a variety ofdifferent geographical locations and appear to be widelydistributed (13-15). Despite their common occurrence in thesubsurface, their numbers are very low at pristine sites, withtypically < 1 to 100 per g being found when they are present(13). Most soil protozoa feed on bacteria; however, at theselow densities they would have little influence on subsurfacebacterial populations. Since the presence of protozoa insubsurface sediments is related to bacterial abundance, itappears that numbers of protozoa are limited by the avail-ability of bacteria to use as a source of nutrition. Bacterialabundance, in turn, is limited by the amount of utilizableorganic carbon present, which, under pristine conditions,decreases below the root zone (22). At sites where thesubsurface is contaminated with utilizable organic contami-nants, bacteria may grow on the contaminants (6). It istherefore likely that subsurface protozoa could be abundantat sites contaminated with organic compounds and may playimportant ecological roles at these sites. Madsen et al. (9)reported large numbers of protozoa at the water table of a

coal tar-contaminated site where oxygen from the unsatur-ated zone mixed with contaminants at the water table.Kinner et al. (8) found elevated numbers of protozoa in a

municipal waste groundwater plume at Cape Cod, Mass.Fuel hydrocarbons are known to be readily biodegraded,

and work by Rogerson and Berger (11) indicated that proto-zoa may be associated with this process. Because of thelikelihood of protozoan activity in the subsurface at a

fuel-contaminated site, we obtained samples from the sur-

face to below the water table at a site having aviationgasoline and jet fuel spills. Our objective was to determinewhether protozoa were numerous enough in the contami-nated areas of the subsurface to play a significant role in themicrobial community. We considered parameters that mightaffect protozoan distribution in contaminated areas. Finally,we collected data on hydraulic conductivity changes during

* Corresponding author.

the course of H202 biotreatment of the aviation gasolinesource area because biomass buildup during this sort oftreatment can reduce hydraulic conductivity. The presence

of protozoa would limit this biomass buildup.

MATERIALS AND METHODS

Field site. The field site was a U.S. Coast Guard AirStation in Traverse City, Mich., where spills of aviationgasoline and jet fuel had occurred. The surface soil of thissite had been frequently disturbed because of constructionand other activities on the Coast Guard base. Sediments inthe study area consisted of sand which exhibited littlevariation in texture or other properties. The water table wasroughly 5.0 m below the surface, but the exact depth variedby more than 1 m depending on weather conditions. Thegeology, hydrology, description of the contaminated areasand of the contaminants, and history of the contaminationproblem are discussed by Twenter et al. (18). The location ofcontaminant source areas, plumes, and sampling locationsare given in Fig. 1.

Samples. Subsurface samples were acquired by using a

drill rig equipped with a hollow-stem auger that was adaptedfor aseptically paring off the outer surface of the core to yielda microbiologically uncontaminated 2-in. (5.08-cm)-diametercore, as was described in Wilson et al. (22). The cores were

pared and the uncontaminated core material was extrudedinto sterile 0.5-pint (ca. 0.2-liter) mason jars in a Plexiglasfield glove box. Samples were immediately placed in an icechest and chilled with ice until they were transferred to a 4°Ccold room at the laboratory within a week.Samples were taken from several boreholes as shown in

Fig. 1. Three categories of areas were sampled, including a

pristine area, parts of contaminated but untreated fuelplumes, and finally, the aviation gasoline source area under-going H202 biotreatment. The site description, boreholedesignation, and date of drilling for boreholes were thefollowing: uncontaminated area, 50CB, 26 June 1990; jet fuelplume, 50CA (approximately 28 m from the jet fuel source),

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468 SINCLAIR ET AL.

InterdictionWells

Smith Hall 5oCc 50OBZ

Aviation Gasoline

50BVParking Lot

SOBX *50CB (Pristine)

Blotreatment Hangar

I

Taxiway 0 50*SOCA Meters

Jet Fuel Plume\

FIG. 1. Locations of boreholes drilled at U.S. Coast Guard AirStation, Traverse City, Mich., for the biotreatment area, the con-taminated but untreated jet fuel and aviation gas plumes, and theuncontaminated area. Borehole 5OBV was drilled on 23 June 1990.Borehole 50BX was drilled on 24 June 1990. Boreholes 50CA and50BZ were drilled on 25 June 1990. Boreholes 50CC and 50CB were

drilled on 26 June 1990. Borehole 50CA was located about 28 m fromthe jet fuel source. Boreholes 5OBV, 50CC, and 50BZ were located85, 257, and 329 m, respectively, from the aviation gasoline source.

25 June 1990; aviation gasoline plume (three boreholes),5OBV on 23 June 1990, 50CC on 26 June 1990, and 50BZ on25 June 1990, which were 85, 257, and 329 m from thesource, respectively; aviation gasoline source area whichwas undergoing hydrogen peroxide biorestoration, 50BX, 24June 1990. The boreholes were drilled 1 to 2 m from soil gasclusters (clusters of tubes for gas sampling extending todifferent depths in the unsaturated zone) so that unsaturatedzone gases could be obtained for all but one of the boreholes(5OBZ). The samples were taken from a profile extendingfrom the unsaturated zone to below the water table. Surfacesoil samples were also taken at the uncontaminated site andthe aviation gasoline source area undergoing biorestoration,but samples were not taken from other boreholes since a

comparison of surface soils and subsurface sediments hasbeen done previously (14, 15).

Field data collection. Water table measurements weremade in the boreholes that were drilled. At the time ofdrilling, soil gas measurements were taken from the soil gasclusters located near the boreholes where samples weretaken. Hydrocarbon vapors, oxygen, and carbon dioxidewere measured with a Bacharach TLV combustible gasdetector and a Bacharach model 302 oxygen indicator. Also

TABLE 1. Data from uncontaminated site 50CB'

Protozoa Bacteria DHA Fuel CSample Depth (m) (g of dw') (CFU g of

Surface 0.00 3.1 x 104 2.3 x 107 BD50CB3 2.21-2.31 1.4 x 102 3.8 x 106 0.11 14.850CB4 3.27-3.37 1.4 x 101 4.7 x 104 0.05 BD50CB10 4.80-4.90 2.5 x 101 7.3 x 106 0.05 BD50CB14 5.31-5.41 <2.1 x 100 7.3 x 106 0.05 BD

a The water table was 5.17 m deep. dw, dry weight.b CFU on PTYG medium. Bacterial counts by AODC were uniform for all

samples, varying between 5.0 x 108 (surface) and 1.3 x 107 (50CB3) of dryweight1.

c BD, below detection.

TABLE 2. Unsaturated zone gases for uncontaminated site 50CB

Depth Fuel vapors % 02 % C02(m) (ppm)1 40a 20.92 40 20.83 40 20.74 40 20.6 0.135 40 20.5a 40 ppm is the normal background level.

at this time, a tracer test was conducted in the aviationgasoline source area, using chloride as a tracer. A pulse ofchloride was injected into the treatment area in the nutrientsolution that was being perfused through the aviation gaso-line spill. Samples of the solution passing through thetreatment area were sampled at different wells located dif-ferent distances from the injection area. Sampling lasteduntil chloride breakthrough occurred at the most distantmonitoring well. Chloride was determined by the mercuricnitrate method (1).

Laboratory analyses. One chemical or biological determi-nation was done per sample. Fuel carbon in sediments wasdetermined by extracting sediments with methylene chlorideand analyzing with a gas chromatograph as described inVandegrift and Kampbell (19). The activity of the soilmicrobial community was measured by assaying dehydroge-nase activity (DHA). Twelve grams of soil was placed in a40-ml bottle. Four milliliters of 0.86% 2,3,5-triphenyltetra-zolium chloride was added, and the sealed bottle was storedat 37°C for 24 h. After incubation, 25 ml of methanol wasadded, and the contents were mixed. The slurry was filteredthrough Whatman no. 5 filter paper, and the absorbance ofthe filtrate was measured with a spectrophotometer set at485 nm. The fuel and DHA analyses were done approxi-mately 1 week after samples were collected. Protozoa weredetermined by a modification of the Singh (16) most-proba-ble-number technique described in Sinclair and Ghiorse (14).Five replicate cultures were used at each dilution level, anddilution levels of 101 to 107 were used. Nongrowing Entero-bacter aerogenes was supplied as a food source. Cultureswere examined by making wet mounts which were examinedunder a phase microscope. Cultures were examined up to 2months after being set up to allow protozoa adequate time toexcyst. This technique was used because it is suited forenumerating protozoa at a broad range of densities down todensities of < 1 per g. Bacteria were enumerated via acridineorange direct counts (AODC) and plate counts, using the

TABLE 3. Data from the jet fuel plume about 28 m fromthe jet fuel source, site 50CA'

Protozoa Bacteria DI-A Fuel CSample Depth (m) (g of dw-1) (CFU g of

50CA2 2.31-2.41 3.7 x 102 1.3 x 107 0.11 71.350CA5 3.05-3.15 3.6 x 104 1.4 x 107 BDc50CA10 4.78-4.88 <1.9 x 10° 0.21 78650CA14 5.36-5.46 2.7 x 101 1.8 x 107 0.21 2,690

a The water table was 5.3 m deep. dw, dry weight.b CFU on PTYG medium. Bacterial counts by AODC were uniform for all

samples, varying between 1.2 x 108 (SOCA10) and 3.3 x 108 (50CA14) g of dryweight - 1.

c BD, below detection.

APPL. ENVIRON. MICROBIOL.

PROTOZOA IN SUBSURFACE OF FUEL SPILL AREAS 469

TABLE 4. Unsaturated zone gases for site 50CA

Depth Fuel vapors % 02 % C02(in) (ppm)1 150 12.0 1.72 180 11.4 1.03 200 7.4 1.74 2,500 4.6 2.2

dilute PTYG medium described in Balkwill and Ghiorse (3)and sample preparations and dilutions as described in Sin-clair and Ghiorse (15). AODC of soils and sediments were

done about 1 week after collection of samples, and bacterialplate counts and protozoan most-probable-number determi-nations were done 1 to 7 months after collection of samples.

RESULTS

Table 1 shows that numbers of protozoa per gram (dryweight) of soil or sediment at the uncontaminated site wereabout the same as those reported for other uncontaminatedsites (15), being in the range of <2 up to 137 per g (dryweight) of sediment. Bacterial numbers were also in a range

that is typical for uncontaminated sites (Table 1). DHAremained constant below 3.27 m. No fuel hydrocarbons weredetected at this site with a field soil gas-measuring instru-ment. A small amount of fuel carbon was detected at 2.2 m.Little oxygen depletion was observed in the unsaturatedzone gases, with 20.9% being present at 1 m and 20.5% beingpresent at 5 m (Table 2). Only one CO2 measurement wasmade in the unsaturated zone (0.13% at 4 m), and thismeasurement indicated that the soil gases included a smallamount of CO2.Table 3 shows data for the jet fuel plume. At 3.05 to 3.15

m, 3.6 x 104 protozoa per g were found, which is equivalentto protozoan numbers normally found in surface soils andvery much higher than typical protozoan densities in thesubsurface. Above and below this depth, protozoan popula-tion densities were much lower. Bacterial numbers as deter-mined by plate counts or AODC did not show any significantdifferences in any of the samples analyzed, although DHAdid increase with depth. Fuel carbon was not detected at3.05 to 3.15 m deep where protozoan activity was greatest;however, fuel carbon was detected above and below thisdepth, with increasing amounts being present at greaterdepths. At 3.0 m deep, where protozoan numbers were

greatest, the fuel vapors were beginning to increase com-

pared with shallower depths (Table 4). However, the 200

TABLE 5. Data from the aviation gasoline plume 85 mfrom the source, site 5OBV"

Protozoa Bacteria DHA Fuel CSample Depth (m) (gotozoa (CFU g of (pg/g)C

(gofdw-1) dw-1)b (Gg/g) g/

5OBV4 2.18-2.28 7.2 x 103 6.7 x 106 0.16 BD5OBV6 3.20-3.30 1.2 x 103 5.1 x 107 0.26 BD50BV20 4.50-4.60 8.1 x 100 2.1 x 107 0.26 BD50BV26 5.28-5.38 <2.1 x 100 9.8 x 106 0.26 5385OBV30 6.02-6.12 1.0 x 104 1.4 x 107 0.11 BD

a The water table was about 5.3 m deep. dw, dry weight.b CFU on PTYG medium. AODC varied between 4.3 x 108 (50BV4) and 8.2

x 108 (50BV30) g of dry weight-'.I BD, below detection.

TABLE 6. Unsaturated zone gases in site 5OBV

Depth Fuel vapors % 02 % C02(in) (ppm)

1 140 10.2 1.32 150 6.8 1.33 220 4.3 1.34 4,100 3.7 1.35 9,500 3.3 1.7

ppm of fuel vapor detected at 3.0 m was much lower thanthat found 1 m deeper, where fuel vapors were present at2,500 ppm. Also at 3 m, oxygen was 7.4% of the soil gas anddeclining with depth. CO2 was 1.7% of unsaturated zone gas

at 3.0 m, and CO2 levels were increasing with depth.Tables 5 through 8 show data for boreholes 5OBV and

50CC drilled in the untreated portions of the aviation gaso-line plume 85 and 257 m, respectively, from the aviationgasoline source. Protozoan numbers in both of these bore-holes showed the same trend noted above for the jet fuelplume. Large protozoan populations were found in some

part of the unsaturated zone, and protozoan numbers de-clined with depth toward the saturated zone where thefloating layer of fuel was found. Another zone of elevatedprotozoan densities was observed just below the water tableand the layer of floating fuel in boreholes 5OBV and 50CC.This zone was detected in 5OBV and 50CC because theseboreholes were sampled from greater depths than borehole50CA in the jet fuel plume. Generally, bacterial numbers didnot show any great changes with depth, although somewhatlower plate counts were noted at site 50CC between 2.31 and4.88 m. DHA followed different trends in these two bore-holes, with the highest activity in the unsaturated zone of5OBV and the highest level of activity being below the watertable at 50CC. Fuel carbon was not detected in any samplesexcept those near the water table at both 5OBV and 50CC.The relation between protozoa and unsaturated soil gases at5OBV followed a trend similar to that seen in the jet fuelplume. Protozoan numbers were highest when fuel vaporswere at 150 ppm, oxygen was at 6.8%, and CO2 was at 1.3%,all of which occurred at 2.0 m. At this depth fuel vapors wereincreasing and the percent oxygen was decreasing withdepth. At site 50CC fuel vapors were much more concen-

trated than at 5OBV, and also the levels of oxygen and CO2indicated that there was reduced biological activity com-

pared with 5OBV.Data for site 50BZ (near the interdiction wells and the

TABLE 7. Data from the aviation gasoline plume 257 mfrom the source, site 50CC'

Protozoa Bacteria DA FeSample Depth (in) Prtza (CFU g of DA Fe(g of dw-1) dw-1)b (pg/g) (plg/g)C

5OCC3 1.32-1.40 6.7 x 103 2.5 x 107 0.11 BD5OCCS 2.31-2.41 <1.9 x 100 3.1 x 106 0.11 BD5OCC7 3.28-3.38 2.0 x 100 4.5 x 105 0.11 BD5OCC13 4.78-4.88 <2.2 x 100 3.5 x 106 0.05 1,4405OCC17 5.33-5.43 2.4 x 104 2.8 x 107 0.11 BD50CC20 6.07-6.17 <2.1 x 100 1.4 x 107 0.16 BD

a The water table was 4.92 m deep. dw, dry weight.b CFU on PTYG medium. AODC varied between 1.2 x 108 (50CC3) and 1.4

x 109 (50CC7) g of dry weight-'.I BD, below detection.

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APPL. ENVIRON. MICROBIOL.

TABLE 8. Unsaturated zone gases in site 50CC

Depth Fuel vapors % 02 % C02(in) (ppm)1 1,200 18.5 0.82 4,600 17.5 0.33 10,000 16.4 0.44 15.6 0.5

north edge of the aviation gasoline plume) are shown inTable 9. Protozoan and bacterial populations as well as DHAactivity from samples collected at this site were not differentfrom those of the uncontaminated site 50CB. Fuel carbonwas not detected.The final category of site sampled was 50BX in the

aviation gasoline source area which was undergoing H202biotreatment (Table 10). Protozoan numbers at 50BX were

much higher than in the contaminated but untreated parts ofthe plumes but followed the same trends observed in thoseareas. At 2.31 to 2.41 m, 3.7 x 105 protozoa per g (dryweight) were found, and protozoan densities declined withdepth to the water table. At a depth of 6.1 to 6.2 m, whichwas below the water table and the floating fuel, protozoannumbers reached their highest level of 6.5 x 105 per g (dryweight). Bacterial numbers as determined by plate countsand AODC showed little change with depth in the subsur-face. DHA increased below 3.28 m. Fuel carbon was onlydetected near the water table in samples from 4.60 to 5.36 mdeep. As was noted in the untreated parts of the aviationgasoline and jet fuel plumes at sites 50CA and 5OBV,protozoan numbers were highest in the unsaturated zone,where the unsaturated zone gases contained 150 ppm of fuelvapors (2 m deep). The oxygen concentration at this depthwas 13.5% and the CO2 level was 0.8% (Table 11).

Flagellates and amoebae were found in roughly equalnumbers in the uncontaminated and the fuel-contaminatedboreholes. A collared flagellate was particularly abundant inthe subsurface at this site. Also, ciliates were found innumbers of 100 or fewer in some samples from the fuel-contaminated boreholes. Ciliates occurred in samples50BX4, -11, -15, and -17 of the H202 biorestoration area. Theonly other sample in which ciliates were present was in50CC3 in the unsaturated zone above a heavily contaminatedarea of the aviation gasoline plume.

Hydraulic conductivity data (data not shown) collectedduring biological treatment of the aviation gasoline sourcearea indicated that little clogging of the aquifer occurredduring introduction of H202 and mineral nutrients. Measure-

TABLE 9. Data from the aviation gasoline plume 329 mfrom the source, site 50BZ'

Protozoa Bacteria DHA Fuel CSample Depth (m) (g of dw-1) (CFU g of Gkg/g) GLg/g),

50BZ2 2.31-2.41 2.4 x 101 6.2 x 106 0.37 BD50BZ4 3.25-3.35 2.5 x 102 1.9 x 106 0.05 BD50BZ11 4.57-4.67 2.3 x 100 3.3 x 106 0.11 BD50BZ15 5.36-5.46 3.7 x 101 1.7 x 106 0.0050BZ17 6.10-6.20 <2.1 x 100 1.8 x 105 0.05 BD

a The water table was 4.53 m deep. dw, dry weight.b CFU on PTYG medium. AODC of bacteria varied between 1.2 x 108

(50BZ15) and 9.2 x 108 (50BZ2) g of dry weight-.c BD, below detection.

TABLE 10. Data from the aviation gasoline source, site 50BX,which was undergoing H202 biorestorationa

Protozoa Bacteria DHA Fuel CSample Depth (in) (g of (CFU g of DA Fe

dw-1) dw- )6 (14gg) (p1g/g)C

Surface 0.00 3.7 x 105 2.4 x 108 11.2150BX2 2.31-2.41 3.7 x 105 5.6 x 106 0.11 BD50BX4 3.28-3.38 1.5 x 104 2.2 x 106 0.05 BD50BX11 4.60-4.70 5.9 x 104 3.6 x 107 0.11 3.550BX15 5.26-5.36 5.8 x 104 2.2 x 107 0.16 43.550BX17 6.10-6.20 6.5 x 105 3.2 x 107 0.16 BD

a The water table at this site was 4.7 m deep. dw, dry weight.b CFU on PTYG medium. The AODC for these samples varied between 3.5

x 108 (5OBX11) and 1.5 x 109 (50BX2) g of dry weight-'.I BD, below detection.

ments of water table differences in wells near the injectionpoint of the nutrients into the biotreatment area indicatedthat there was a 20% decrease in hydraulic conductivity inJune 1990 after treatment compared with March 1988 beforetreatment. Also, Fig. 2 shows the results of chloride tracertests in which chloride was pumped through the treatmentarea before and after treatment. The breakthrough of thechloride was slightly retarded (<20%) in June 1990 aftertreatment compared with March 1988 before treatment.

DISCUSSION

These results show that protozoa can become very numer-ous in the subsurface at fuel-contaminated sites. The areas ofgreatest protozoan abundance were in the unsaturated zone,where fuel vapors mixed with atmospheric oxygen, andslightly below the layer of floating fuel on the water table,presumably where oxygenated groundwater came into con-tact with the fuel. Dissolved oxygen in groundwater slightlybelow the layer of floating fuel in the aviation gasoline sourcewas found to be 3 ppm by Wilson et al. (sample R2 in Table1 of reference 21). In the unsaturated zone of fuel-contami-nated sites 50CA, 50BX, and 5OBV, protozoa reached theirhighest numbers when fuel vapors were in the range of 150 to200 ppm. Other characteristics of this region of high proto-zoan abundance were that oxygen levels were declining withdepth and CO2 levels were elevated compared with shal-lower depths. The exception was site 50CC, where fuelvapors were at 1,200 ppm at 1 m and protozoan numberswere elevated at 1.32 m. Site 50CC may not be comparableto the other sites because the fuel vapor concentration wasmuch higher than at the other contaminated sites. Also, thesmall changes of oxygen and CO2 levels with depth suggestthat less biological activity was occurring in the subsurfaceof 50CC compared with 50CA and 50BX. Increased proto-zoan activity could be observed in the saturated zone belowthe layer of floating fuel at sites 50BX, 5OBV, and 50CC,where oxygenated groundwater came into contact with the

TABLE 11. Unsaturated zone gases in the site undergoingH202 biorestoration (site 50BX)

Depth Fuel vapors % 02 % C02(in) (ppm)

1 90 19.0 0.72 150 13.5 0.83 1,600 9.0 2.74 4,100 5.0 0.7

470 SINCLAIR ET AL.

PROTOZOA IN SUBSURFACE OF FUEL SPILL AREAS 471

1.1 will develop in enrichment cultures because of culturalo 1.0 DG-31-2 * t'i * conditions, food incompatibilities, or other problems. Singhc 0.9 * 0 1 (16) found that about 70% of added protozoa were recovered

*|@ ** by using the most-probable-number technique outlined in his0.8 paper, and Foissner (5) reviewed papers which reported a0.7 * 0 much lower recovery for some protozoan groups with the

O(O same technique. Therefore, it must be assumed that most-v 0.6 probable-number enumeration methods will underestimate° 0.5 the number of protozoa present. Sinclair and Ghiorse (14)

5 ° reported that encysted protozoan numbers in one sample, 0.4 were not distinguishable from total protozoan numbers.

.0 0.3 X°0 Therefore, it is likely that the numbers of protozoa reported02 *Test 1 in this study do not represent active forms. Nonetheless,

E 0*2 ° oTet 2 despite enumeration problems and problems with extrapo-z 0.1 0 Oo lating activity from only numbers, the large numbers of

_, protozoa reported in this work compared to numbers re-0.0 ported in uncontaminated subsurface sediments suggest that

0 50 100 150 200 250 considerable protozoan growth had occurred.

Elapsed Time (hours) The protozoan populations found in the H202 biorestora-FIG. 2. Chloride tracer test breakthrough curve for chloride tion area were as large or larger than those found in the

introduced into the injection well in the biotreatment area and surface soil samples examined in this study and conse-monitored in a well 8.4 m away. The data are for the most quently could be expected to significantly reduce bacterialcontaminated interval, which was slightly below the water table. populations. The observed bacterial population in sampleThe solid circles represent tracer test 1, which was done in March 50BX from the aviation gasoline biotreatment area was 7.0 x1988 before treatment, and the open circles represent a tracer test 108 cells per g (dry weight) as counted by AODC, which isdone in June 1990 after about 2 years of treatment. substantially less than would be expected to have grown

from an observed value of 6 mg of fuel (as determined by ouranalyses) degraded per g of sediment for this depth. The

fuel. This effect was accentuated at site 50BX, where oxygen protozoan population in the same sample was 6.5 x 105/gfrom H202 and also mineral nutrients were present in the (dry weight). Reports, including those by Anderson et al. (2)groundwater. and Clarholm (4) among others, indicate that a protozoan

Protozoan populations appeared to be more sensitive to population of this size can have a major impact on theoxygen levels than bacteria. Protozoa were most abundant in bacterial population in soil.areas of the unsaturated zone, which contained at least 6% Bioremediation of subsurface sediments is dependent on aoxygen, and beneath the floating fuel, where oxygenated sufficient hydraulic conductivity to permit pumping nutrientsgroundwater was present. By contrast, bacteria seemed to through the affected area. Bacterial growth in porous mediaadapt to local conditions and showed less change in numbers when abundant nutrients are present has often been ob-in different parts of the profile than protozoa did. This was served to cause large reductions in hydraulic conductivity (7,illustrated in earlier unpublished work in which a transition 10, 12, 20). Taylor and Jaffd (17) reported that hydraulicwas observed in bacterial types from strict aerobes to conductivity in soil columns was reduced by a factor of 10 tofacultative anaerobes with increasing depth at the jet fuel- >1,000 when 1.0 to 1.2 mg of bacterial organic carbon percontaminated area. cm3 (0.5 to 0.6 mg of bacterial organic carbon per g of

In addition to adaptation to anaerobic conditions, the lack sediment, assuming the sediment had a specific gravity ofof any great change in bacterial numbers in different samples 2.0) of soil grew from methanol. In the biotreatment area, 6through the profile may have also been due to a lack of mg/g of fuel was degraded. Assuming that the bacteriaprotozoan grazing at certain depths which allowed slowly degrading fuel had a 50% growth efficiency, 3 mg of bacterialgrowing bacteria to accumulate. Therefore, bacterial popu- carbon per g of sediment would have been produced. There-lation sizes alone would not necessarily be an indicator of fore, enough bacterial carbon may have been producedbacterial activity. The DHA measurements also suggest that during biotreatment in the aviation gasoline source area toactivity and bacterial numbers are not always related, as can reduce hydraulic conductivity in some cases (if the bacteriabe seen in DHA and bacterial plate counts in borehole 50CC. made an extracellular capsule, as presumably happened inNo elevated protozoan populations were observed at the methanol-fed soil columns of Taylor and Jaffe [17]).

borehole 50BZ, which was located near the edge of where Despite the bacterial growth which occurred in thethe aviation gasoline plume had been and farther from the biotreatment area, hydraulic conductivity did not decreasesource area than the other boreholes. When the same significantly. The hydraulic conductivity, as measured bylocation was sampled on 4 August 1989, 1.7 x 105 protozoa head differences in wells in the treatment area, was reducedper g of dry weight were found in a sample from 4.65 to 5.25 by about 20% between March 1988, before treatment began,m deep, which would be in the saturated zone near the water and June 1990, when treatment ended. Supporting thistable. Apparently, between August 1989 and June 1990, observation is the chloride tracer data of Fig. 2, whichchanges occurred in both the levels of fuel and the microbial indicate that the hydraulic conductivity was reduced bypopulations present in the distant parts of the plume. <20% after treatment in the aviation gasoline source area.

Most-probable-number enrichment culture techniques to Therefore, circumstantial evidence is presented that proto-enumerate protozoa such as what was used in this study zoa may play a role in maintaining hydraulic conductivityhave inherent limitations to their accuracy, as has been during biotreatment of readily degraded organic contami-discussed by Foissner (5). Not all protozoa in a soil sample nants. Further work will be needed to quantify the effect of

VOL. 59, 1993

472 SINCLAIR ET AL.

reduction of bacterial populations by protozoan grazing onhydraulic conductivity in sediments.

ACKNOWLEDGMENTS

We thank Lowell Leach, Monty Frazier, Alton Tweedy, TimHensley, and Frank Beck of the drill crew for assistance withdrilling and sampling. We also thank Jim Weaver for assisting withinterpretation of hydraulic conductivity data and Chris Griffin forproviding site information.

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the examination of water and wastewater, 17th ed., p. 4-69,4-70. American Public Health Association, Washington, D.C.

2. Anderson, R. V., E. T. Elliot, J. F. McClellan, D. C. Coleman,C. V. Cole, and H. W. Hunt. 1978. Trophic interactions in soilsas they affect energy and nutrient dynamics. III. Biotic interac-tions of bacteria, amoebae, and nematodes. Microb. Ecol.4:361-371.

3. Balkwill, D. L., and W. C. Ghiorse. 1985. Characterization ofsubsurface bacteria associated with two shallow aquifers inOklahoma. Appl. Environ. Microbiol. 50:580-588.

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