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Time-dependent changes in viable numbers and activities of aerobic heterotrophic bacteria in subsurface samples
JOURNAL OF ELSEVIER
Journal
of Microbiological
Methods
21 (1995)
253-265
Time-dependent changes in viable numbers and activities of aerobic heterotrophic bacteria in subsurface samples J.K.
Fredricksona’*,
S.W. Li”, F.J. Brockmana, P.S. Amyb, D.L. Balkwill’
D.L.
Haldemanb,
“Pacific Northwest Laboratory, K4-06, P.O. Box 999, Richland, WA 99352, USA ‘University of Nevada Las Vegas, Las Vegas, NE 89154, LISA ‘Florida State University, Tallahassee, FL 32308, USA Received
17 February
1994; revision
received
28 May 1994;
; accepted
30 May 1994
Abstract
Vadose and saturated zone sediment cores from depths to 212 m were obtained from the U.S. Department of Energy’s Hanford Site in south-central Washington by cable tool drilling, and volcanic ashfall tuff samples were obtained from tunnels 400 m beneath the surface of Rainier Mesa at DOE’s Nevada Test Site (NTS) in southern Nevada. Numbers of viable aerobic heterotrophic bacteria were determined by plate counts and metabolic activities were determined by [‘4C]glucose radiorespirometry at t = 0 and at various post-sampling time points up to 154 d in order to assess the influence of sample storage on microbiological properties of subsurface samples. Increases in post-sampling populations of viable bacteria were observed in all samples, although the magnitude of the increase and time after sampling at which the maximum population size was reached varied with the sample type. The greatest post-sampling increases in viable counts and [‘4C]glucose mineralization occurred in a high-organic carbon lacustrine sediment. The population of aerobic heterotrophs increased from below detection at t = 0 to > lo6 CFU g-’ after 139 d. Significant increases in culturable counts were shown to occur within 24 h for tuff samples from NTS. These results indicate that precautions are necessary in the post-drilling handling of subsurface sediments and rock for microbiological analysis. In addition, these results suggest that even low biomass subsurface environments may be readily stimulated for applications such as in situ bioremediation. Keywords:
Storage; Subsurface;
* Corresponding author. [email protected].
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0167-7012/95/$09.50 @ 1995 Elsevier SSDI 0167-7012(94)00053-O
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254
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Fredrickson
et al.
I Journal of Microbiological Methods 21 (1995) 253-265
1. Introduction
Although significant strides have been made over the past several decades, the study of subsurface microbiology is still hindered by the inaccessibility of the subsurface and the associated difficulty of obtaining representative samples. It is generally accepted, although little definitive information is available, that groundwater samples obtained from wells have microbiological characteristics distinct from sediment or rock samples obtained from the same formation by coring. This has been attributed to the predominance of attached microorganisms in subsurface microbial communities [7]. Kolbel-Boelke et al. [9] found that numbers of bacteria based on viable plate counts and direct microscopic count methods were lo- to l,OOO-fold higher in subsurface sediments than in groundwater and that the composition of the microbial community was quite different in the two types of subsurface samples. Therefore, it has been general practice to consider core material as the representative sample for microbiological investigations. An added complication is that the drill sites from which core samples are obtained are often remote, requiring the transport of samples to distant laboratories for analyses. This generally implies that periods ranging from hours to days may elapse between the time that samples are collected and the time that analyses are initiated. There is currently little information with regard to the influence of the time elapsed between sampling and analyses on the microbiological properties of subsurface samples. Hirsch and Rades-Rohkohl [8] found that the number of viable cells in groundwater samples increased with time up to 9 days, observing increases in counts as high as 440-fold. This phenomenon was also associated with changes in the proportion of pigmented Gram-negative bacteria, indicating shifts in composition of the microbial community. In vadose zone samples that were stored at 4°C for between 75 and 97 days (d), numbers of viable bacteria increased as much as l,OOO-fold, while there was little or no increase in the number of total bacteria as determined by direct microscopic counting [3]. The results suggest that significant changes in the microbiological properties of subsurface sediments can take place over time. It is generally accepted that environmental materials sampled for microbiological study need to be analyzed as soon as possible or preserved in order to obtain representative results. However, there is little information available identifying and quantifying the microbiological changes that take place in environmental materials following sampling. The purpose of these studies was to investigate the influence of storage time on the microbiological properties of saturated and unsaturated subsurface sediments.
2. Materials and methods 2.1. Description of sampling sites Core samples were collected from the vadose and saturated zones on the U.S. Department of Energy’s Hanford Site in south-central Washington at depths
J.K.
Fredrickson
et al. I Journal of Microbiological
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255
ranging from 24 to 212 m. Cores were obtained from two boreholes, one of which, 299-W18-246 (W18), was located within an area that had been impacted by artificial recharge water or contaminants from past site activities [4]. One borehole, termed the Yakima Barricade (YB), was located approximately 7 km from the other borehole and was hydrologically upgradient from areas of the Hanford Site affected by past site operations. Samples of volcanic ashfall tuff were obtained from tunnels within the Rainier Mesa on the U.S. Department of Energy’s Nevada Test Site (NTS) in southern Nevada. The depths below the surface from which the core or rock samples were obtained and a description of the samples are presented in Table 1. Hanford Site samples were obtained from a sequence of glaciofluvial deposits informally referred to as the Hanford formation and from the Ringold Formation, fluviallacustrine deposits from the ancestral Columbia and Salmon-Clearwater Rivers. The first two samples, 47.0 and 71.7 m, from the YBB were from the vadose zone, and the other four were from the saturated zone. Underlying the Hanford formation at the W18 site are two strata not present at the YBB. Volcanic ashfall tuff samples were obtained from the U12n tunnel, a portion of the 26 km of mined tunnels, located 400 m beneath Rainier Mesa on the NTS. 2.2. Sampling methods All subsurface samples from the Hanford Site were obtained using cable-tool percussion drilling with a split-spoon core barrel. Cable tool drilling method was selected for coring subsurface sediments at Hanford because it does not require the use of circulating drilling fluids. The split-spoon core barrel was 60 cm long and 10 cm in diameter and contained a Lexan liner that had been sterilized by autoclaving. Procedures used for disinfection of sampling tools and processing of samples were similar to those described elsewhere [lo]. For the vadose zone samples, dry tracer mix containing l-pm-diameter carboxylated YG fluorescent microspheres (Polysciences, Warrington, PA) (5 ml) as a microbiological tracer was prepared and introduced to the bottom of the YBB using a bailer just prior to coring, as described by Fredrickson et al. [4]. For the saturated zone samples, microspheres in a Whirlpak bag were placed in the shoe just in front of the core catcher so that the bag would rupture, releasing the microspheres, as it was pushed through the prongs by the core. Following coring, the Lexan liner containing the core was immediately removed from the split-spoon core barrel and the exposed ends were capped. Cores were transported to facilities where they were carefully opened in an Ar- or N,-filled glovebag. Using sterile tools, sediment from the center of the cores was carefully removed for microbiological, chemical, and physical analyses. Sampling in this manner resulted in disaggregation of semiconsolidated sediments or rock and essentially complete mixing in the case of unconsolidated, coarse-grained sediments. Microspheres in the sampled microscopy; the lower core material were enumerated usin g epifluorescent detection limit was lo3 microspheres g- . No tracers were used during drilling and sampling of the W18 borehole. Two samples from the YBB, 187.6 and 211.5 m, had low concentrations of microspheres even after paring (Table 1).
Rainier Mesa
h nd = not done.
Nevada Test Site
’ bd = below detection:
Early “Palouse” Plio-Pleistocene
sot1
Unit
flood hands
developed)
developed)
sands developed)
reolitized
ashfall tuff
paleosol (weakly developed) calcrete (strongly developed)
cataclysmic
cataclysmic flood paleosol (weakly lacustrine paleosol (strongly fluvial sand paleosol (strongly
Lithology
4no
24.4 43.6 447
47.0 71.7 174.x 187 6 IY‘t I 211.5
(m)
Sample depth
nd
nd nd
ndh
750 680 CSOO
nd
x0
hd 3.3
[email protected] 230 9610 2090
(mg kg’)
Total organic C
hd” bd bd 3.x
Microspheres (log g ~’ dry wt.)
2.7 9.8 11.5
20.0
22.2
4.8 2.8 2Y.6 36.1
Watercontent (% dry wt. basis)
B P 2 F;.
2
??
%
$ a a,
formation
z
Hanford
Test
HanfordlWlX
State and Nevada
formation Formatton Formation Formation Formation Formation
Washington
Hanford Ringold Ringold Ringold Ringold Ringold
Site in south-central
HanfordlYBB
Hanford
.
of Energy’s
%
from the LIS Department
Stratigraphic
samples obtained
SiteiBorehole
subsurface
Characteristics of vadose and saturated Site in southern Nevada
Table I
J.K.
Fredrickson
et al. I Journal of Microbiological
Methods 21 (1995) 253-265
257
Samples from the W18 borehole and the two vadose zone samples from YBB were maintained at 4”C, whereas samples from the saturated zone were stored at 17°C the approximate in situ temperature. Tuff samples from the NTS were obtained from fresh rock faces using aseptic procedures as previously described [6]. Additional treatments were imposed upon the tuff samples to investigate the influence of post-sampling storage temperature and physical disruption of rock fragments on culturable counts and colony morphotype distribution. Intact tuff fragments, 5-10 g in size, were homogenized by grinding with a sterile mortar and pestle and placed in sterile, airtight containers that were maintained at -20, 4, and 24°C. Culturable counts were determined on subsamples that were removed from each of these treatments at various post-sampling times. Care was taken at each sampling point to prevent contamination of sediment or rock. 2.3. Viable counts Populations of viable aerobic heterotrophic bacteria in the Hanford samples, expressed as colony-forming units (CFU) per gram of dry sediment, were determined by blending samples in 0.1% Na,P,O, . lOH,O at pH 7.0, then spread-plating lo-fold serial dilutions of sediment in phosphate-buffered saline in triplicate on 1% peptone-tryptone-yeast extract-glucose agar (PTYG) [2,5] or optimal plate count agar (OPCA) (Stevens, submitted for publication). OPCA was developed to optimize the culture of subsurface aerobic heterotrophic bacteria and was used for spread plate enumeration (vs. pour plate enumeration) in this study. OPCA contains per liter, 0.25 g KH*PO,, 0.4 g K,HPO,, 0.5 g NH,Cl, 0.02 g MgCl, * 6H,O, 1.0 g glucose, 0.05 g peptone, 0.05 g tryptone, 0.1 g yeast extract, and 15 g of agar. In addition, one ml of a trace metals solution containing per 100 ml, 1.5 g CaCl, *2H,O, 0.7 g FeSO, .7H,O, 0.5 g Na,SO,, 0.5 g MnCl, .4H,O, 0.05 g H,BO,, 0.05 g ZnCl,, 0.05 g CoCl, 36H,O, 0.05 g NiCl, - 6H,O, 0.03 g CuClz .2H,O, and 0.01 g Na,MoO, - 2H,O was added to each liter of OPCA. One ml of a vitamin solution that contained per 100 ml, 0.01 g of vitamin B,, 0.01 g riboflavin, 0.01 g thiamine, 0.01 g of vitamin B12, 0.005 g of nicotinamide 0.005 g of p-aminobenzoic acid, 0.006 g of lipoic acid, 0.002 g biotin, and 0.002 g folicin was also added to each liter. Finally, 1.0 g of autoclaved activated charcoal was added to the sterile medium. Plates were incubated for 2-3 weeks at 22°C and the number of bacterial colonies on appropriate dilutions was determined. Viable counts in the tuff samples from NTS were determined on R2A agar (Difco), as previously described [1,6]. One of each distinct colony morphotype was selected from dilution plates that were enumerated for the various tuff samples and treatments. Colony morphotypes [1,5] were described and recorded for most of the samples and colonies representative of each morphotype from the 187.6-m YBB core sample at t = 0 and at different post-sampling times were subcultured and purified. API Rapid NFT kits (Analytab Products, Plainview, NY) were used to characterize these isolates for 21 physiological traits.
258
.I. K. Fredrickson
2.4. [“C]Glucose
et al.
i Journal of Microbiological Methods 21 (1995) 253-265
mineralization
The ability of the indigenous heterotrophic population to metabolize carbon substrates was determined by measuring the mineralization of 0.25 PCi of [U-‘4C]glucose (3 PCi prnol-I) purchased from NEN Research Products (Boston, MA). The total volume of solution added to 10 g of sediment was 200 ~1 so as to minimize the change in sediment water potential [3]. For the YBB saturated samples, 5 g of sediment and 100 ~1 of the labeled substrate were used. Bottles were incubated in the dark at 22°C. Evolved 14C02 was trapped in 1 ml of 1 N KOH in liquid-scintillation vials suspended in the headspace. The amount of 14C0, evolved over time was measured by liquid scintillation counting (Wallac model 1411, Pharmacia LKB, Gaithersburg, MD). The cumulative percent of ]‘4C]glucose mineralized was plotted against sampling time and regression analysis was performed using the initial linear portion of the plot. Mineralization rates were determined as the slope (% d-r) obtained from linear regression. Controls consisted of sand, which was autoclaved on three consecutive days and treated in a manner identical to the subsurface samples. Mineralization of [14C]glucose in the sterile sand controls was negligible (0.01% d-’ ? 1 SD).
3. Results 3.1. Viable counts Although the response varied with the individual sampie, the number of viable aerobic heterotrophic bacteria in all the subsurface samples increased with prolonged storage (Table 2). The increases in viable counts were negligible in the YBB vadose samples from 47.0 and 71.7 m. However, increases from below detection (approximately 10 CFU g-‘) to lo6 CFU g-’ in the YB saturated zone samples from 174.8 m and lower were observed. While most of the viable counts were low or below detection at t = 0, the W18 sample from 43.6 m was quite high at 6.6 log CFU g-‘, probably due to stimulation by water and/or contaminants [4]. Despite relatively high numbers of culturable bacteria in that sample, there was a significant increase in number, reaching to 8.1 log CFU g-’ over a 20 d post-sampling period. In general, the maximum viable counts in the Hanford subsurface samples were not attained until 21 d, and in some cases 70 d, post-sampling. In addition, in samples from 174.8, 187.6, and 211.5 m the elevated numbers tended to remain high for extended periods with little or no decline. A rapid increase was also observed in the population of culturable aerobic heterotrophic bacteria in the homogenized tuff samples maintained at 24°C (Fig. 1). Viable counts also increased in the homogenized tuff maintained at 4°C although the lag time was greater than for the tuff incubated at 24°C. There was an increase in viable counts in the intact fragments stored at 4”C, although the lag time was even longer and the response was not as great in magnitude as with the
J. K. Fredrickson
et al. i Journal of Microbiological
Table 2 Populations of culturable aerobic heterotrophic time post-sampling Borehole
Sample depth (m)
YBB
47.0
71.7
174.8
187.6
194.1
211.5
W18
24.4 43.6 44.7
Methods 21 (1995) 253-265
259
bacteria (viable counts) in subsurface samples with No. of distinct colony morphotypes
Post-sampling time (d)
Viable counts (log CFU g-‘) 1% PTYG
OPCA
1 18 47 70 147 1 15 42 84 133 0 9 22 77 139 0 6 21 70 144 0 21 63 137 0 38 91 154
nd” nd nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd nd nd 1 nd 8 14 16 2 3 3 8 31. 0 17 nd 19 0 2 11 22
0 37 0 20 0 21
nd nd nd nd nd nd
nd nd nd nd nd nd
a nd = not done.
homogenized tuff. There was no change in viable counts in the homogenized frozen at - 20°C. 3.2. Colony morphotypes
tuff
and API types
Selection of representative colonies by morphotype is relatively effective for identifying organisms with similar physiology and fatty acid methyl ester profiles
260
J.K.
Fredrickson
et ul.
/ Journal of Microbiological Methods 21 (195)
253-265
1
lE+031”.‘,““,“‘.:.“.) 0
----Q..-
Homo. -20°C
----A-----
Intact, 4°C
-...__._ 10
20
30
40
Time, d
Fig. 1. Changes in viable counts of aerobic heterotrophic with increasing post-sampling time. Tuff was homogenized or was kept as intact fragments (intact) at 4°C.
bacteria in volcanic ashfall tuff from NTS (homo) and maintained at 24, 4 or -2O”C,
[5] and hence was used to assess changes in the types of bacteria that were recovered on agar medium at the various time points among the different treatments. In the YB saturated zone samples, the number of distinct colony morphotypes increased with increasing post-sampling time (Table 2), paralleling the increases in viable counts. Analyses of colony morphotypes from NTS tuff samples at t = 0 and at various post-sampling times indicated that between 19% and 35% of the morphotypes were recovered only within the period between t = 0 and 24 h after sampling (Table 3). Approximately 16-34% of unique morphotypes were also recovered only at the sampling points between 7 and 30 d. Similar results were obtained for isolates obtained at various post-sampling time points from the Hanford paleosol sample using API rapid NIT kits (Table 4). In general, greater than 35% of the isolates obtained at one time point had a unique NFT profile. However, some types were recovered from each time point including one tentatively identified as Pseudomonas stuzeri by virtue of very good or excellent matches.
Table 3 Distribution
of unique
colony
Sample treatment
Homogenized tuff (4°C) Homogenized tuff (24°C) Homogenized tuff (-20°C) Intact fragments (4°C)
morphotypes
in NTS tuff samples
No. morphotypes
at various
at time t/no.
morphotypes
t=Oto24h
r=3to5d
24169 27182 12/64 18159
21/68 33183 42164 24159
(35%) (33%) (19%) (31%)
post-sampling
times
at all times t=7to30d
(31%) (27%) (65%) (40%)
23168 (34%) 22/82(270/o) 10164 (16%) 17/59(29%)
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Table 4 Distribution of unique API types in Hanford subsurface paleosol sample from 187.6 m stored at 17°C and sampled and plated at various times Time of plating (d) 0
7 21 70 144
No. distinct API types/ no. distinct colony morphotypes
No. API types observed at time t only
212 313 313 7/S 24131
1 3 2 3 19
3.3. Substrate mineralization The metabolic activities of heterotrophic microorganisms, as measured by the rate of [‘4C]glucose mineralization, in the impacted vadose samples from the W18 borehole were high at t = 0, and there was little or no increase after 20-37 d post-sampling (Fig. 2a-c). Rates of [ 14C]glucose mineralization in unimpacted vadose sediments from the YBB were very low or below detection immediately after sampling (t = 0) but increased significantly after extended (70-147 d) post-sampling periods (Fig. 2d-e). In the saturated samples from the YBB, glucose mineralization rates were low immediately after sampling (Fig. 2f-i), but with post-sampling times of 21 d or greater there were dramatic increases in [ 14C]glucose mineralization rates.
4. Discussion There was a consistent time-dependent increase in the number of culturable aerobic heterotrophic bacteria in all of the subsurface samples examined in this study. The rate and magnitude of the increases in population size varied for each sample and, for the tuff, were also dependent on post-sampling storage temperature and whether fragments were homogenized or left intact. In the unimpacted Hanford Site vadose zone sediments, which were deemed microbially impoverished [4], the increases in viable counts were modest as was the stimulation of [‘4C]glucose mineralization. In contrast, the increase in viable counts in the unimpacted 174.8-m sample from the saturated lacustrine strata took them from below detection at t = 0 to greater than lo6 CFU g-’ after 77 d at 17°C. Concomitant with the increase in viable counts was an increase in the glucose mineralization rate, from 1.8% to 6.0% d-l. Even in those samples in which viable populations and activities were relatively high at t = 0, such as in the impacted Hanford vadose samples from the W18 borehole and in the NTS tuff samples, increases in post-sampling viable counts were substantial. One possible explanation for the post-sampling increases in viable counts and
262
J. K. Fredrickson et al. I Journal of Microbiological Methods 21 (1995) 253-265
Post-sampling time, d Fig. 2. Rates of [ 14C]glucose mineralization in subsamples of Hanford subsurface sediments (y-axis) amended with the labeled substrate at various post-sampling times as indicated on the x-axis. Bars with the same hatch patterns indicate that the samples came from the same borehole.
activity is the outgrowth of bacterial contaminants introduced during drilling and sampling. This is unlikely for several reasons. First, latex microspheres used as a microbial tracer during drilling operations were below detection in 4 of the 6 samples where they were used, and the post-sampling microbial changes were no greater or more rapid than in those samples where they were detected. In addition, these results are consistent with those presented earlier for subsurface vadose sediments from the Hanford Site in which viable counts and glucose mineralization rate constants increased after extended post-sampling periods [3]. Several of the vadose zone samples used in that study and those from the NTS were sampled from outcrops or mined tunnels where the potential for contamination due to sampling is much less than when drilling and coring are required. Although major increases in viable counts in most of the Hanford Site subsurface sediments were not apparent until several weeks after sampling, increases were usually detectable within 6 to 9 days. In the tuff samples from
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NTS, increases in viable counts of up to lOO-fold occurred within as little as 24 h. Hirsch and Rades-Rohkohl [8] observed 17-fold increases in viable bacteria counts and a loss of diversity in groundwater samples stored for 24 h. Keeping temperatures cool and limiting disruption of rock fragments or sediments appeared to delay, but did not prevent, an increase in viable counts. This indicates that maintaining cool temperatures and preventing physical disruption prior to initiating microbiological analyses of subsurface samples are necessary to ensure results that are representative of in situ characteristics. It also suggests that critical analyses should be initiated as soon as possible after sampling, preferably within 24 h. The effects of sample storage on the activities and populations of soil microorganisms have been evaluated by several investigators. West et al. [13] found that glucose-induced microbial respiration rates in arable and grassland soils decreased with storage at 25°C over a period of two to ten weeks. There was also a general decline in bacterial and mycelial volumes over this same period. Unfortunately, there was no data on the microbiological properties of the soil samples used in that study from time points earlier than two weeks. However, in an earlier study, West et al. [12] found that biomass-C decreased within 7 d after sampling while ATP concentrations actually increased. In experiments to examine the effects of longer storage periods, up to 56 d, on microbial biomass, Ross et al. [ 111 found the effects of storage at 4°C or 25°C to be variable and dependent upon soil type with both increases and decreases in biomass observed upon storage. Although the effects of storage on soil microbial biomass and activity appear to vary with soil type, there is a general trend for decreasing biomass and activity with time upon storage. These results contrast with the findings of this study that show essentially the opposite effect for subsurface sediment and rock. These differences are likely related to the fact that surface soils generally contain large populations of diverse, active microorganisms whereas uncontaminated subsurface environments, particularly deep vadose sediments [4], are nutrient impoverished. Several intriguing questions regarding post-sampling increases in viable counts and activity arise as the result of these findings. Are the responses observed in subsurface materials the result of growth of a few starved or dormant cells, i.e., r-strategists, or the result of a more general resuscitation of many inactive cells? What are the mechanisms involved in triggering these responses? Although it is not possible to rigorously address these questions using the results presented herein, some insights can be gained. Between 16% and 65% of the individual colony morphotypes were obtained within only one of the three sampling intervals (Table 3) and 35% or greater of the API NFT types were obtained at only one of five different sampling times (Table 4). In a study by Brockman et al. [3] of vadose zone sediments from Hanford, significant increases in viable counts occurred with increasing post-sampling time, although populations determined by acridine orange direct counts did not change over the same time interval. These results suggest that the post-sampling increases in viable counts observed in subsurface samples are not simply due to outgrowth of a few cells. Differences in
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community composition could be due to a variety of complex factors, including different lag times for resuscitation of various bacteria, growth and succession, and even the heterogeneous distribution of microorganisms. The fact that distinct colony morphotypes are obtained after different periods of storage (Table 3) suggests that at least part of the variation in the types of organisms obtained at the different times is due to heterogeneous distribution of microorganisms in samples. Both Brockman et al. [3] and Hirsch and Rades-Rohkohl [S] reported significant changes in community composition and decreases in microbial diversity with storage of vadose and groundwater samples, respectively. The factors responsible for these changes deserve increased attention in the future. The differences in viable counts between the intact and homogenized tuff fragments may provide some insight into the potential mechanisms accounting for post-sampling stimulation of viable counts and activities. The increase in viable counts of heterotrophic bacteria in the tuff was more rapid and extensive in the homogenized samples than in the intact fragments. Similar results have been obtained with vadose and saturated subsurface sediments from the Hanford Site (F. Brockman and J. Fredrickson, unpublished data). This suggests that there may be a redistribution of nutrients, moisture, or O2 that increases their availability to microorganisms, as a result of disaggregation and physical mixing. Studies with saturated subsurface samples from the eastern coastal plain and from the Hanford Site (T. Phelps and F. Brockman, unpublished data) showed that addition of sterile deionized water resulted in significant increases in microbial activity, probably the result of an increase in nutrient availability. Substantial increases in viable counts occurred even in intact tuff fragments, but the increase was lo-fold less than when they were homogenized. In the intact fragments, redistribution of nutrients as the result of mixing would be reduced. It would be interesting to determine if increases in viable counts occurred primarily at or near the exposed surfaces or if an increase in viable bacteria was distributed more evenly throughout the fragments. The results presented herein demonstrate that there is a general increase in the numbers and types of viable heterotrophic bacteria and metabolic activity following sampling of subsurface sediments and rock. This phenomenon occurred regardless of whether subsurface samples were left intact or were homogenized. These results suggest that precautions must be taken during sampling and processing of subsurface materials to limit the amount of time before starting analyses and experiments. Alternatively, such increases may lend insight into the types of microorganisms that may be stimulated by contamination or recharge or by in situ manipulation for bioremediation.
Acknowledgements
We wish to thank Bruce Bjornstad and Llyn Doremus for their assistance in obtaining the Hanford Site core samples used in this study. This research was supported by the Subsurface Science Program, Office of Health and Environmen-
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265
tal Research, U.S. Department of Energy (DOE). The continued support of Dr. F.J. Wobber is greatly appreciated. Pacific Northwest Laboratory is operated for the DOE by Battelle Memorial Institute under Contract DE-AC06-76RL0 1830. The Nevada Test Site research is supported by grant DOE-UNLV-60975 to the University of Nevada, Las Vegas.
References [l] Amy, P.S., Haldeman, D.L., Ringelberg, D., Hall, D.H. and Russell, C. (1992) Comparison of identification systems for classification of bacteria isolated from water and endolithic habitats within the deep subsurface. Appl. Environ. Microbial. 58, 3367-3373. [2] Balkwill, D.L. (1989) Numbers, diversity, and morphological characteristics of aerobic. chemoheterotrophic bacteria in deep subsurface sediments from a site in South Carolina. Geomicrobiol. J. 7, 33-51. [3] Brockman, F.J., Kieft, T.L., Fredrickson, J.K., Bjornstad, B.N., Li, S.W., Spangenburg, W. and Long, P.E. (1992) Microbiology of vadose zone paleosols in South-central Washington state. Microb. Ecol. 23, 279-301. [4] Fredrickson, J.K., Brockman, F.J., Bjornstad, B.N.. Long, P.E., Li, S.W., McKinley. J.P., Wright, J.V., Conca, J.L., Kieft, T.L. and Balkwill. D.L. (1993) Microbiological characteristics of pristine and contaminated deep vadose sediments from an arid region. Geomicrobiol. J. 11, 95-107. [5] Haldeman, D.L. and Amy, P.S. (1993) Diversity within a colony morphotype: implications for ecological research. Appl. Environ. Microbial. 59, 933-935. [6] Haldeman, D.L., Amy, P.S., Ringelberg, D. and White, D.C. (1993) Characterization of the microbiology within a 21 m3 section of rock from the deep subsurface. Microb. Ecol. 26. 145-159. [7] Hazen, T.C., Jimenez, L., de Victoria, G.L. and Fliermans, C.B. (1991) Comparison of bacteria from deep subsurface sediment and adjacent groundwater. Microb. Ecol. 22, 293-304. [8] Hirsch. P. and Rades-Rohkohl, E. (1988) Some special problems in the determination of viable counts of groundwater microorganisms. Microb. Ecol. 16, 99-113. [9] Kdlbel-Boelke, J., Anders, E.-M. and Nehrkorn, A. (1988) Microbial communities in the saturated groundwater environment II: diversity of bacterial communities in a Pleistocene sand aquifer and their in vitro activities. Microb. Ecol. 16, 31-48. [lo] Phelps, T.J., Fliermans, C.B., Garland, T.R., Pfiffner, S.M. and White, D.C. (1989) Recovery of deep subsurface sediments for microbiological studies. J. Microbial. Methods 9, 267-280. (111 Ross, D.J., Tate, K.R., Cairns, A. and Meyrick, K.F. (1980) Influence of storage on soil microbial biomass estimated by three biochemical procedures. Soil Biol. Biochem. 12, 369-374. [12] West, A.W., Ross, D.J. and Cowling, J.C. (1986) Changes in microbial C, N, P and ATP contents, numbers and respiration on storage of soil. Soil Biol. Biochem. 18, 141-148. [13] West, A.W., Ross, D.J. and Cowling, J.C. (1987) Relationships between mycelial and bacterial populations in stored, air-dried and glucose-amended arable and grassland soils. Soil Biol. Biochem. 19. 599-605.
Journal
of Microbiological
Methods
21 (1995)
253-265
Time-dependent changes in viable numbers and activities of aerobic heterotrophic bacteria in subsurface samples J.K.
Fredricksona’*,
S.W. Li”, F.J. Brockmana, P.S. Amyb, D.L. Balkwill’
D.L.
Haldemanb,
“Pacific Northwest Laboratory, K4-06, P.O. Box 999, Richland, WA 99352, USA ‘University of Nevada Las Vegas, Las Vegas, NE 89154, LISA ‘Florida State University, Tallahassee, FL 32308, USA Received
17 February
1994; revision
received
28 May 1994;
; accepted
30 May 1994
Abstract
Vadose and saturated zone sediment cores from depths to 212 m were obtained from the U.S. Department of Energy’s Hanford Site in south-central Washington by cable tool drilling, and volcanic ashfall tuff samples were obtained from tunnels 400 m beneath the surface of Rainier Mesa at DOE’s Nevada Test Site (NTS) in southern Nevada. Numbers of viable aerobic heterotrophic bacteria were determined by plate counts and metabolic activities were determined by [‘4C]glucose radiorespirometry at t = 0 and at various post-sampling time points up to 154 d in order to assess the influence of sample storage on microbiological properties of subsurface samples. Increases in post-sampling populations of viable bacteria were observed in all samples, although the magnitude of the increase and time after sampling at which the maximum population size was reached varied with the sample type. The greatest post-sampling increases in viable counts and [‘4C]glucose mineralization occurred in a high-organic carbon lacustrine sediment. The population of aerobic heterotrophs increased from below detection at t = 0 to > lo6 CFU g-’ after 139 d. Significant increases in culturable counts were shown to occur within 24 h for tuff samples from NTS. These results indicate that precautions are necessary in the post-drilling handling of subsurface sediments and rock for microbiological analysis. In addition, these results suggest that even low biomass subsurface environments may be readily stimulated for applications such as in situ bioremediation. Keywords:
Storage; Subsurface;
* Corresponding author. [email protected].
Tel.:
0167-7012/95/$09.50 @ 1995 Elsevier SSDI 0167-7012(94)00053-O
Heterotrophs;
+ 1
(509)
Science
Tuff; Lacustrine;
375-3908.
B.V. All rights
Fax:
reserved
+ 1
Vadose
(509)
375-6666.
E-mail:
254
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Fredrickson
et al.
I Journal of Microbiological Methods 21 (1995) 253-265
1. Introduction
Although significant strides have been made over the past several decades, the study of subsurface microbiology is still hindered by the inaccessibility of the subsurface and the associated difficulty of obtaining representative samples. It is generally accepted, although little definitive information is available, that groundwater samples obtained from wells have microbiological characteristics distinct from sediment or rock samples obtained from the same formation by coring. This has been attributed to the predominance of attached microorganisms in subsurface microbial communities [7]. Kolbel-Boelke et al. [9] found that numbers of bacteria based on viable plate counts and direct microscopic count methods were lo- to l,OOO-fold higher in subsurface sediments than in groundwater and that the composition of the microbial community was quite different in the two types of subsurface samples. Therefore, it has been general practice to consider core material as the representative sample for microbiological investigations. An added complication is that the drill sites from which core samples are obtained are often remote, requiring the transport of samples to distant laboratories for analyses. This generally implies that periods ranging from hours to days may elapse between the time that samples are collected and the time that analyses are initiated. There is currently little information with regard to the influence of the time elapsed between sampling and analyses on the microbiological properties of subsurface samples. Hirsch and Rades-Rohkohl [8] found that the number of viable cells in groundwater samples increased with time up to 9 days, observing increases in counts as high as 440-fold. This phenomenon was also associated with changes in the proportion of pigmented Gram-negative bacteria, indicating shifts in composition of the microbial community. In vadose zone samples that were stored at 4°C for between 75 and 97 days (d), numbers of viable bacteria increased as much as l,OOO-fold, while there was little or no increase in the number of total bacteria as determined by direct microscopic counting [3]. The results suggest that significant changes in the microbiological properties of subsurface sediments can take place over time. It is generally accepted that environmental materials sampled for microbiological study need to be analyzed as soon as possible or preserved in order to obtain representative results. However, there is little information available identifying and quantifying the microbiological changes that take place in environmental materials following sampling. The purpose of these studies was to investigate the influence of storage time on the microbiological properties of saturated and unsaturated subsurface sediments.
2. Materials and methods 2.1. Description of sampling sites Core samples were collected from the vadose and saturated zones on the U.S. Department of Energy’s Hanford Site in south-central Washington at depths
J.K.
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255
ranging from 24 to 212 m. Cores were obtained from two boreholes, one of which, 299-W18-246 (W18), was located within an area that had been impacted by artificial recharge water or contaminants from past site activities [4]. One borehole, termed the Yakima Barricade (YB), was located approximately 7 km from the other borehole and was hydrologically upgradient from areas of the Hanford Site affected by past site operations. Samples of volcanic ashfall tuff were obtained from tunnels within the Rainier Mesa on the U.S. Department of Energy’s Nevada Test Site (NTS) in southern Nevada. The depths below the surface from which the core or rock samples were obtained and a description of the samples are presented in Table 1. Hanford Site samples were obtained from a sequence of glaciofluvial deposits informally referred to as the Hanford formation and from the Ringold Formation, fluviallacustrine deposits from the ancestral Columbia and Salmon-Clearwater Rivers. The first two samples, 47.0 and 71.7 m, from the YBB were from the vadose zone, and the other four were from the saturated zone. Underlying the Hanford formation at the W18 site are two strata not present at the YBB. Volcanic ashfall tuff samples were obtained from the U12n tunnel, a portion of the 26 km of mined tunnels, located 400 m beneath Rainier Mesa on the NTS. 2.2. Sampling methods All subsurface samples from the Hanford Site were obtained using cable-tool percussion drilling with a split-spoon core barrel. Cable tool drilling method was selected for coring subsurface sediments at Hanford because it does not require the use of circulating drilling fluids. The split-spoon core barrel was 60 cm long and 10 cm in diameter and contained a Lexan liner that had been sterilized by autoclaving. Procedures used for disinfection of sampling tools and processing of samples were similar to those described elsewhere [lo]. For the vadose zone samples, dry tracer mix containing l-pm-diameter carboxylated YG fluorescent microspheres (Polysciences, Warrington, PA) (5 ml) as a microbiological tracer was prepared and introduced to the bottom of the YBB using a bailer just prior to coring, as described by Fredrickson et al. [4]. For the saturated zone samples, microspheres in a Whirlpak bag were placed in the shoe just in front of the core catcher so that the bag would rupture, releasing the microspheres, as it was pushed through the prongs by the core. Following coring, the Lexan liner containing the core was immediately removed from the split-spoon core barrel and the exposed ends were capped. Cores were transported to facilities where they were carefully opened in an Ar- or N,-filled glovebag. Using sterile tools, sediment from the center of the cores was carefully removed for microbiological, chemical, and physical analyses. Sampling in this manner resulted in disaggregation of semiconsolidated sediments or rock and essentially complete mixing in the case of unconsolidated, coarse-grained sediments. Microspheres in the sampled microscopy; the lower core material were enumerated usin g epifluorescent detection limit was lo3 microspheres g- . No tracers were used during drilling and sampling of the W18 borehole. Two samples from the YBB, 187.6 and 211.5 m, had low concentrations of microspheres even after paring (Table 1).
Rainier Mesa
h nd = not done.
Nevada Test Site
’ bd = below detection:
Early “Palouse” Plio-Pleistocene
sot1
Unit
flood hands
developed)
developed)
sands developed)
reolitized
ashfall tuff
paleosol (weakly developed) calcrete (strongly developed)
cataclysmic
cataclysmic flood paleosol (weakly lacustrine paleosol (strongly fluvial sand paleosol (strongly
Lithology
4no
24.4 43.6 447
47.0 71.7 174.x 187 6 IY‘t I 211.5
(m)
Sample depth
nd
nd nd
ndh
750 680 CSOO
nd
x0
hd 3.3
[email protected] 230 9610 2090
(mg kg’)
Total organic C
hd” bd bd 3.x
Microspheres (log g ~’ dry wt.)
2.7 9.8 11.5
20.0
22.2
4.8 2.8 2Y.6 36.1
Watercontent (% dry wt. basis)
B P 2 F;.
2
??
%
$ a a,
formation
z
Hanford
Test
HanfordlWlX
State and Nevada
formation Formatton Formation Formation Formation Formation
Washington
Hanford Ringold Ringold Ringold Ringold Ringold
Site in south-central
HanfordlYBB
Hanford
.
of Energy’s
%
from the LIS Department
Stratigraphic
samples obtained
SiteiBorehole
subsurface
Characteristics of vadose and saturated Site in southern Nevada
Table I
J.K.
Fredrickson
et al. I Journal of Microbiological
Methods 21 (1995) 253-265
257
Samples from the W18 borehole and the two vadose zone samples from YBB were maintained at 4”C, whereas samples from the saturated zone were stored at 17°C the approximate in situ temperature. Tuff samples from the NTS were obtained from fresh rock faces using aseptic procedures as previously described [6]. Additional treatments were imposed upon the tuff samples to investigate the influence of post-sampling storage temperature and physical disruption of rock fragments on culturable counts and colony morphotype distribution. Intact tuff fragments, 5-10 g in size, were homogenized by grinding with a sterile mortar and pestle and placed in sterile, airtight containers that were maintained at -20, 4, and 24°C. Culturable counts were determined on subsamples that were removed from each of these treatments at various post-sampling times. Care was taken at each sampling point to prevent contamination of sediment or rock. 2.3. Viable counts Populations of viable aerobic heterotrophic bacteria in the Hanford samples, expressed as colony-forming units (CFU) per gram of dry sediment, were determined by blending samples in 0.1% Na,P,O, . lOH,O at pH 7.0, then spread-plating lo-fold serial dilutions of sediment in phosphate-buffered saline in triplicate on 1% peptone-tryptone-yeast extract-glucose agar (PTYG) [2,5] or optimal plate count agar (OPCA) (Stevens, submitted for publication). OPCA was developed to optimize the culture of subsurface aerobic heterotrophic bacteria and was used for spread plate enumeration (vs. pour plate enumeration) in this study. OPCA contains per liter, 0.25 g KH*PO,, 0.4 g K,HPO,, 0.5 g NH,Cl, 0.02 g MgCl, * 6H,O, 1.0 g glucose, 0.05 g peptone, 0.05 g tryptone, 0.1 g yeast extract, and 15 g of agar. In addition, one ml of a trace metals solution containing per 100 ml, 1.5 g CaCl, *2H,O, 0.7 g FeSO, .7H,O, 0.5 g Na,SO,, 0.5 g MnCl, .4H,O, 0.05 g H,BO,, 0.05 g ZnCl,, 0.05 g CoCl, 36H,O, 0.05 g NiCl, - 6H,O, 0.03 g CuClz .2H,O, and 0.01 g Na,MoO, - 2H,O was added to each liter of OPCA. One ml of a vitamin solution that contained per 100 ml, 0.01 g of vitamin B,, 0.01 g riboflavin, 0.01 g thiamine, 0.01 g of vitamin B12, 0.005 g of nicotinamide 0.005 g of p-aminobenzoic acid, 0.006 g of lipoic acid, 0.002 g biotin, and 0.002 g folicin was also added to each liter. Finally, 1.0 g of autoclaved activated charcoal was added to the sterile medium. Plates were incubated for 2-3 weeks at 22°C and the number of bacterial colonies on appropriate dilutions was determined. Viable counts in the tuff samples from NTS were determined on R2A agar (Difco), as previously described [1,6]. One of each distinct colony morphotype was selected from dilution plates that were enumerated for the various tuff samples and treatments. Colony morphotypes [1,5] were described and recorded for most of the samples and colonies representative of each morphotype from the 187.6-m YBB core sample at t = 0 and at different post-sampling times were subcultured and purified. API Rapid NFT kits (Analytab Products, Plainview, NY) were used to characterize these isolates for 21 physiological traits.
258
.I. K. Fredrickson
2.4. [“C]Glucose
et al.
i Journal of Microbiological Methods 21 (1995) 253-265
mineralization
The ability of the indigenous heterotrophic population to metabolize carbon substrates was determined by measuring the mineralization of 0.25 PCi of [U-‘4C]glucose (3 PCi prnol-I) purchased from NEN Research Products (Boston, MA). The total volume of solution added to 10 g of sediment was 200 ~1 so as to minimize the change in sediment water potential [3]. For the YBB saturated samples, 5 g of sediment and 100 ~1 of the labeled substrate were used. Bottles were incubated in the dark at 22°C. Evolved 14C02 was trapped in 1 ml of 1 N KOH in liquid-scintillation vials suspended in the headspace. The amount of 14C0, evolved over time was measured by liquid scintillation counting (Wallac model 1411, Pharmacia LKB, Gaithersburg, MD). The cumulative percent of ]‘4C]glucose mineralized was plotted against sampling time and regression analysis was performed using the initial linear portion of the plot. Mineralization rates were determined as the slope (% d-r) obtained from linear regression. Controls consisted of sand, which was autoclaved on three consecutive days and treated in a manner identical to the subsurface samples. Mineralization of [14C]glucose in the sterile sand controls was negligible (0.01% d-’ ? 1 SD).
3. Results 3.1. Viable counts Although the response varied with the individual sampie, the number of viable aerobic heterotrophic bacteria in all the subsurface samples increased with prolonged storage (Table 2). The increases in viable counts were negligible in the YBB vadose samples from 47.0 and 71.7 m. However, increases from below detection (approximately 10 CFU g-‘) to lo6 CFU g-’ in the YB saturated zone samples from 174.8 m and lower were observed. While most of the viable counts were low or below detection at t = 0, the W18 sample from 43.6 m was quite high at 6.6 log CFU g-‘, probably due to stimulation by water and/or contaminants [4]. Despite relatively high numbers of culturable bacteria in that sample, there was a significant increase in number, reaching to 8.1 log CFU g-’ over a 20 d post-sampling period. In general, the maximum viable counts in the Hanford subsurface samples were not attained until 21 d, and in some cases 70 d, post-sampling. In addition, in samples from 174.8, 187.6, and 211.5 m the elevated numbers tended to remain high for extended periods with little or no decline. A rapid increase was also observed in the population of culturable aerobic heterotrophic bacteria in the homogenized tuff samples maintained at 24°C (Fig. 1). Viable counts also increased in the homogenized tuff maintained at 4°C although the lag time was greater than for the tuff incubated at 24°C. There was an increase in viable counts in the intact fragments stored at 4”C, although the lag time was even longer and the response was not as great in magnitude as with the
J. K. Fredrickson
et al. i Journal of Microbiological
Table 2 Populations of culturable aerobic heterotrophic time post-sampling Borehole
Sample depth (m)
YBB
47.0
71.7
174.8
187.6
194.1
211.5
W18
24.4 43.6 44.7
Methods 21 (1995) 253-265
259
bacteria (viable counts) in subsurface samples with No. of distinct colony morphotypes
Post-sampling time (d)
Viable counts (log CFU g-‘) 1% PTYG
OPCA
1 18 47 70 147 1 15 42 84 133 0 9 22 77 139 0 6 21 70 144 0 21 63 137 0 38 91 154
nd” nd nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd nd nd 1 nd 8 14 16 2 3 3 8 31. 0 17 nd 19 0 2 11 22
0 37 0 20 0 21
nd nd nd nd nd nd
nd nd nd nd nd nd
a nd = not done.
homogenized tuff. There was no change in viable counts in the homogenized frozen at - 20°C. 3.2. Colony morphotypes
tuff
and API types
Selection of representative colonies by morphotype is relatively effective for identifying organisms with similar physiology and fatty acid methyl ester profiles
260
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Fredrickson
et ul.
/ Journal of Microbiological Methods 21 (195)
253-265
1
lE+031”.‘,““,“‘.:.“.) 0
----Q..-
Homo. -20°C
----A-----
Intact, 4°C
-...__._ 10
20
30
40
Time, d
Fig. 1. Changes in viable counts of aerobic heterotrophic with increasing post-sampling time. Tuff was homogenized or was kept as intact fragments (intact) at 4°C.
bacteria in volcanic ashfall tuff from NTS (homo) and maintained at 24, 4 or -2O”C,
[5] and hence was used to assess changes in the types of bacteria that were recovered on agar medium at the various time points among the different treatments. In the YB saturated zone samples, the number of distinct colony morphotypes increased with increasing post-sampling time (Table 2), paralleling the increases in viable counts. Analyses of colony morphotypes from NTS tuff samples at t = 0 and at various post-sampling times indicated that between 19% and 35% of the morphotypes were recovered only within the period between t = 0 and 24 h after sampling (Table 3). Approximately 16-34% of unique morphotypes were also recovered only at the sampling points between 7 and 30 d. Similar results were obtained for isolates obtained at various post-sampling time points from the Hanford paleosol sample using API rapid NIT kits (Table 4). In general, greater than 35% of the isolates obtained at one time point had a unique NFT profile. However, some types were recovered from each time point including one tentatively identified as Pseudomonas stuzeri by virtue of very good or excellent matches.
Table 3 Distribution
of unique
colony
Sample treatment
Homogenized tuff (4°C) Homogenized tuff (24°C) Homogenized tuff (-20°C) Intact fragments (4°C)
morphotypes
in NTS tuff samples
No. morphotypes
at various
at time t/no.
morphotypes
t=Oto24h
r=3to5d
24169 27182 12/64 18159
21/68 33183 42164 24159
(35%) (33%) (19%) (31%)
post-sampling
times
at all times t=7to30d
(31%) (27%) (65%) (40%)
23168 (34%) 22/82(270/o) 10164 (16%) 17/59(29%)
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Table 4 Distribution of unique API types in Hanford subsurface paleosol sample from 187.6 m stored at 17°C and sampled and plated at various times Time of plating (d) 0
7 21 70 144
No. distinct API types/ no. distinct colony morphotypes
No. API types observed at time t only
212 313 313 7/S 24131
1 3 2 3 19
3.3. Substrate mineralization The metabolic activities of heterotrophic microorganisms, as measured by the rate of [‘4C]glucose mineralization, in the impacted vadose samples from the W18 borehole were high at t = 0, and there was little or no increase after 20-37 d post-sampling (Fig. 2a-c). Rates of [ 14C]glucose mineralization in unimpacted vadose sediments from the YBB were very low or below detection immediately after sampling (t = 0) but increased significantly after extended (70-147 d) post-sampling periods (Fig. 2d-e). In the saturated samples from the YBB, glucose mineralization rates were low immediately after sampling (Fig. 2f-i), but with post-sampling times of 21 d or greater there were dramatic increases in [ 14C]glucose mineralization rates.
4. Discussion There was a consistent time-dependent increase in the number of culturable aerobic heterotrophic bacteria in all of the subsurface samples examined in this study. The rate and magnitude of the increases in population size varied for each sample and, for the tuff, were also dependent on post-sampling storage temperature and whether fragments were homogenized or left intact. In the unimpacted Hanford Site vadose zone sediments, which were deemed microbially impoverished [4], the increases in viable counts were modest as was the stimulation of [‘4C]glucose mineralization. In contrast, the increase in viable counts in the unimpacted 174.8-m sample from the saturated lacustrine strata took them from below detection at t = 0 to greater than lo6 CFU g-’ after 77 d at 17°C. Concomitant with the increase in viable counts was an increase in the glucose mineralization rate, from 1.8% to 6.0% d-l. Even in those samples in which viable populations and activities were relatively high at t = 0, such as in the impacted Hanford vadose samples from the W18 borehole and in the NTS tuff samples, increases in post-sampling viable counts were substantial. One possible explanation for the post-sampling increases in viable counts and
262
J. K. Fredrickson et al. I Journal of Microbiological Methods 21 (1995) 253-265
Post-sampling time, d Fig. 2. Rates of [ 14C]glucose mineralization in subsamples of Hanford subsurface sediments (y-axis) amended with the labeled substrate at various post-sampling times as indicated on the x-axis. Bars with the same hatch patterns indicate that the samples came from the same borehole.
activity is the outgrowth of bacterial contaminants introduced during drilling and sampling. This is unlikely for several reasons. First, latex microspheres used as a microbial tracer during drilling operations were below detection in 4 of the 6 samples where they were used, and the post-sampling microbial changes were no greater or more rapid than in those samples where they were detected. In addition, these results are consistent with those presented earlier for subsurface vadose sediments from the Hanford Site in which viable counts and glucose mineralization rate constants increased after extended post-sampling periods [3]. Several of the vadose zone samples used in that study and those from the NTS were sampled from outcrops or mined tunnels where the potential for contamination due to sampling is much less than when drilling and coring are required. Although major increases in viable counts in most of the Hanford Site subsurface sediments were not apparent until several weeks after sampling, increases were usually detectable within 6 to 9 days. In the tuff samples from
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NTS, increases in viable counts of up to lOO-fold occurred within as little as 24 h. Hirsch and Rades-Rohkohl [8] observed 17-fold increases in viable bacteria counts and a loss of diversity in groundwater samples stored for 24 h. Keeping temperatures cool and limiting disruption of rock fragments or sediments appeared to delay, but did not prevent, an increase in viable counts. This indicates that maintaining cool temperatures and preventing physical disruption prior to initiating microbiological analyses of subsurface samples are necessary to ensure results that are representative of in situ characteristics. It also suggests that critical analyses should be initiated as soon as possible after sampling, preferably within 24 h. The effects of sample storage on the activities and populations of soil microorganisms have been evaluated by several investigators. West et al. [13] found that glucose-induced microbial respiration rates in arable and grassland soils decreased with storage at 25°C over a period of two to ten weeks. There was also a general decline in bacterial and mycelial volumes over this same period. Unfortunately, there was no data on the microbiological properties of the soil samples used in that study from time points earlier than two weeks. However, in an earlier study, West et al. [12] found that biomass-C decreased within 7 d after sampling while ATP concentrations actually increased. In experiments to examine the effects of longer storage periods, up to 56 d, on microbial biomass, Ross et al. [ 111 found the effects of storage at 4°C or 25°C to be variable and dependent upon soil type with both increases and decreases in biomass observed upon storage. Although the effects of storage on soil microbial biomass and activity appear to vary with soil type, there is a general trend for decreasing biomass and activity with time upon storage. These results contrast with the findings of this study that show essentially the opposite effect for subsurface sediment and rock. These differences are likely related to the fact that surface soils generally contain large populations of diverse, active microorganisms whereas uncontaminated subsurface environments, particularly deep vadose sediments [4], are nutrient impoverished. Several intriguing questions regarding post-sampling increases in viable counts and activity arise as the result of these findings. Are the responses observed in subsurface materials the result of growth of a few starved or dormant cells, i.e., r-strategists, or the result of a more general resuscitation of many inactive cells? What are the mechanisms involved in triggering these responses? Although it is not possible to rigorously address these questions using the results presented herein, some insights can be gained. Between 16% and 65% of the individual colony morphotypes were obtained within only one of the three sampling intervals (Table 3) and 35% or greater of the API NFT types were obtained at only one of five different sampling times (Table 4). In a study by Brockman et al. [3] of vadose zone sediments from Hanford, significant increases in viable counts occurred with increasing post-sampling time, although populations determined by acridine orange direct counts did not change over the same time interval. These results suggest that the post-sampling increases in viable counts observed in subsurface samples are not simply due to outgrowth of a few cells. Differences in
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community composition could be due to a variety of complex factors, including different lag times for resuscitation of various bacteria, growth and succession, and even the heterogeneous distribution of microorganisms. The fact that distinct colony morphotypes are obtained after different periods of storage (Table 3) suggests that at least part of the variation in the types of organisms obtained at the different times is due to heterogeneous distribution of microorganisms in samples. Both Brockman et al. [3] and Hirsch and Rades-Rohkohl [S] reported significant changes in community composition and decreases in microbial diversity with storage of vadose and groundwater samples, respectively. The factors responsible for these changes deserve increased attention in the future. The differences in viable counts between the intact and homogenized tuff fragments may provide some insight into the potential mechanisms accounting for post-sampling stimulation of viable counts and activities. The increase in viable counts of heterotrophic bacteria in the tuff was more rapid and extensive in the homogenized samples than in the intact fragments. Similar results have been obtained with vadose and saturated subsurface sediments from the Hanford Site (F. Brockman and J. Fredrickson, unpublished data). This suggests that there may be a redistribution of nutrients, moisture, or O2 that increases their availability to microorganisms, as a result of disaggregation and physical mixing. Studies with saturated subsurface samples from the eastern coastal plain and from the Hanford Site (T. Phelps and F. Brockman, unpublished data) showed that addition of sterile deionized water resulted in significant increases in microbial activity, probably the result of an increase in nutrient availability. Substantial increases in viable counts occurred even in intact tuff fragments, but the increase was lo-fold less than when they were homogenized. In the intact fragments, redistribution of nutrients as the result of mixing would be reduced. It would be interesting to determine if increases in viable counts occurred primarily at or near the exposed surfaces or if an increase in viable bacteria was distributed more evenly throughout the fragments. The results presented herein demonstrate that there is a general increase in the numbers and types of viable heterotrophic bacteria and metabolic activity following sampling of subsurface sediments and rock. This phenomenon occurred regardless of whether subsurface samples were left intact or were homogenized. These results suggest that precautions must be taken during sampling and processing of subsurface materials to limit the amount of time before starting analyses and experiments. Alternatively, such increases may lend insight into the types of microorganisms that may be stimulated by contamination or recharge or by in situ manipulation for bioremediation.
Acknowledgements
We wish to thank Bruce Bjornstad and Llyn Doremus for their assistance in obtaining the Hanford Site core samples used in this study. This research was supported by the Subsurface Science Program, Office of Health and Environmen-
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tal Research, U.S. Department of Energy (DOE). The continued support of Dr. F.J. Wobber is greatly appreciated. Pacific Northwest Laboratory is operated for the DOE by Battelle Memorial Institute under Contract DE-AC06-76RL0 1830. The Nevada Test Site research is supported by grant DOE-UNLV-60975 to the University of Nevada, Las Vegas.
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