Abstract
Environmental conditions which define boundaries for biofilm production could provide useful ecological information for biofilm models. A practical use of defined conditions could be applied to the high-level nuclear waste repository at Yucca Mountain. Data for temperature and humidity conditions indicate that decreases in relative humidity or increased temperature severely affect biofilm formation on three candidate canister metals.
Biofilms create microenvironments for a consortium of bacteria residing on a substrate. These microenvironments include variations in pH, nutrient concentrations, and oxygen levels (17, 24). On a surface such as metal, biofilms allow for a variety of microorganisms with differing redox potential requirements to reside in close proximity. Microorganisms that carry out microbially influenced corrosion (MIC) can occur in and are facilitated by biofilms (7, 8, 9). MIC also includes production of microbial metabolites at one location which diffuse to a corrosion site, possibly at another location (15). MIC of metal surfaces results in pitting, crevice corrosion, under-deposit corrosion, and selective leaching. The ability of endolithic and contaminating microbes to form biofilms can ultimately affect performance of structures such as those in the Yucca Mountain repository.
Metal biocorrosion occurs in the presence of biofilms; therefore, this study was designed to determine boundary conditions for biofilm formation. Our study had two objectives that address the potential for biofilm production: (i) to determine relative humidity (RH) limits for biofilm formation, and (ii) to determine the boundary limits of biofilm formation on the basis of temperature.
Yucca Mountain is located at the Nevada Test Site, 100 miles northwest of Las Vegas, Nevada. Mined Yucca Mountain tuff was collected near the north portal entrance. The surface layer of the tunnel wall was removed by using flame-sterilized tools, and the newly exposed rock was collected into sterile, plastic bags and was placed on ice. The rock was transported to the laboratory within 6 h and was stored at −20°C (14). It was later aseptically crushed into fine grains by using flame-sterilized mortars and pestles.
One-centimeter-square coupons were constructed from the following metals: C22 nickel alloy, N-316 stainless steel, and titanium (Metal Samples, Mumford, Ala.). Aseptically crushed rock was transferred to sterile glass petri plates, metal coupons were placed within the crushed rock, and microcosms were placed in chambers held to specific RH and temperature values.
To achieve different RH levels, specific saturated salt solutions were placed in the bottom reservoirs of the microcosm chambers (Nalgene Autoclavable Desiccators, Rochester, N.Y.). The salt solutions included KCl (83.6% RH), KI (67.9% RH), and MgCl2 (32.4% RH). Distilled water was used to create 100% RH. Petri plate microcosms were placed above the salt solution reservoir. Chambers were sealed with high-vacuum grease (Dow Corning, Midland, Mich.), and RH levels were checked by using a digital thermohygrometer (Cole-Palmer P-37450-52; Vernon Hills, Ill.). Microcosms were equilibrated for 1 week prior to addition of metal coupons.
Selected temperatures were achieved by using a Revco incubator (model RA50-1060-ABA; Asheville, N.C.) for 30°C, a Napco incubator (model 303; Winchester, Pa.) for 60°C, and a Precision Scientific Thelco laboratory oven (model 160; Winchester, Pa.) for 70°C. National Institute of Standards and Technology-traceable thermometers (H-B Instruments, Trappe, Pa.) were used to monitor temperatures.
At designated time points, adherent material and biofilm was scraped off coupons and was homogenized by vortexing in 1.0 ml of R2 broth (1). Homogenized biofilm (100 μl) was spread plated onto R2A agar (Difco/BD Diagnostics Systems, Sparks, Md.) and were incubated at room temperature (25°C) for 2 weeks. CFU were counted, and microbial diversity was calculated by using the Shannon-Weaver diversity index (2).
For scanning electron microscopy (SEM), coupons were immersed in 4% glutaraldehyde (Sigma, St. Louis, Mo.) for 18 h and were allowed to air dry for an additional 24 h. Coupons prepared for SEM (Jeol JSM-5600 SEM; Peabody, Mass.) were gold coated by using a Pelco model 3 sputter coater. Microscopy was conducted at the University of Nevada, Las Vegas (EPMA/SEM Facility). Coupon samples were analyzed at days 0 and 1 and at months 1, 5, 12, and 18. Images for 12- and 18-month incubations appeared very similar for all metal coupon types and environmental conditions.
RH effects on biofilm formation.
At 100% RH and 30°C the heterotrophic plate count (HPC) values from all three metal types increased from day 0 to day 1 from less than 1 × 102 to 2 × 104 to 4 × 104 CFU per coupon (Tables 1, 2, and 3). All three metal types supported increased HPC values by 5 months (4 × 104 to 7 × 104 CFU per coupon); however, after 18 months of incubation fewer microbes were cultured (5 × 102 to 8 × 102 CFU per coupon).
TABLE 1.
HPC from N-316 stainless steel coupons over 18 months of incubation at 30°C
| Incubation period | HPC at % RHa:
|
|||
|---|---|---|---|---|
| 100 | 84 | 70.5 | 32 | |
| 0 days | 1.06 × 102 (2.7 × 101) | 1.06 × 102 (2.7 × 101) | 1.06 × 102 (2.7 × 101) | 1.06 × 102 (2.7 × 101) |
| 1 day | 1.88 × 104 (2.18 × 103) | 84 (20) | 32 (8) | 1.90 × 102 (1.15 × 102) |
| 1 mo | 3.88 × 104 (5.91 × 103) | 97 (28) | 0 | 38 (6) |
| 5 mo | 4.25 × 104 (3.30 × 103) | 0 | 7 (3) | 3.52 × 102 (7.20 × 101) |
| 12 mo | 8.78 × 103 (1.89 × 103) | 3 (2) | 1 (1) | 58 (12) |
| 18 mo | 7.61 × 102 (1.57 × 102) | 2.90 × 102 (4.70 × 101) | 1.11 (1.11) | 2.51 × 102 (2.60 × 101) |
Standard error values were calculated from six replicates and are given in parentheses. All values were calculated from triplicate coupons.
TABLE 2.
HPC from C22 nickel alloy coupons over 18 months of incubation at 30°C
| Incubation period | HPC at % RHa:
|
|||
|---|---|---|---|---|
| 100 | 84 | 70.5 | 32 | |
| 0 days | 53 (12) | 53 (12) | 53 (12) | 53 (12) |
| 1 day | 2.98 × 104 (1.80 × 103) | 2.10 × 102 (7.3 × 101) | 22 (4) | 52 (11) |
| 1 mo | 3.22 × 103 (8.30 × 102) | 32 (10) | 0 | 92 (14) |
| 5 mo | 6.73 × 104 (1.23 × 104) | 17 (8) | 30 (11) | 2.88 × 103 (3.10 × 101) |
| 12 mo | 4.88 × 103 (1.47 × 103) | 8 (3) | 0 | 43 (6) |
| 18 mo | 5.04 × 102 (1.00 × 102) | 3.16 × 102 (4.70 × 101) | 0 | 2.51 × 102 (2.60 × 101) |
Standard error values were calculated from six replicates and are given in parentheses. All values were calculated from triplicate coupons.
TABLE 3.
HPC from titanium coupons over 18 months of incubation at 30°C
| Incubation period | HPC at % RHa:
|
|||
|---|---|---|---|---|
| 100 | 84 | 70.5 | 32 | |
| 0 days | 56 (12) | 56 (12) | 56 (12) | 56 (12) |
| 1 day | 3.65 × 104 (4.51 × 103) | 75 (21) | 20 (11) | 1.06 × 102 (2.3 × 101) |
| 1 mo | 5.54 × 104 (1.31 × 104) | 88 (36) | 1 (1) | 87 (8) |
| 5 mo | 4.86 × 104 (1.90 × 104) | 0 | 17 (6) | 9.28 × 102 (1.70 × 102) |
| 12 mo | 1.22 × 104 (1.37 × 103) | 26 (15) | 0 | 1.33 × 102 (2.9 × 101) |
| 18 mo | 7.56 × 102 (8.60 × 101) | 3.37 × 102 (5.40 × 101) | 0 | 2.46 × 102 (2.20 × 101) |
Standard error values were calculated from six replicates and are given in parentheses. All values were calculated from triplicate coupons.
HPC values at 84% RH were lower than 100% RH for all three metal types after day 0 (Tables 1, 2, and 3). Although there was attachment of microorganisms, approximately 2 × 102 CFU per coupon at day 1 for all three coupon types, it was followed by stable to decreasing values until 18 months of incubation, where all three metal coupon types supported increased biofilm development. Before 18 months, each coupon type supported less than detectable numbers of bacteria (102 CFU per coupon) at some time point, but then biofilm development reestablished, nearly equaling the 18-month value at 100% RH.
At 70.5% RH, HPC values consistently decreased from slightly less than 102 CFU per coupon at day 0 for the remainder of the test.
For 32% RH, N-316 coupons demonstrated microbial attachment, 102 CFU per coupon at day 0, which gradually increased and then remained stable during the remainder of the experiment. Titanium coupons showed microbial attachment, less than 102 CFU per coupon at days 0, with an increase in CFU values to almost 103 CFU per coupon after 5 months of incubation; however, by 12 to 18 months of incubation, CFU values decreased by 10-fold to near their original level. In the case of C22 nickel alloy, biofilm production mimicked that of all three coupon types at 100% RH. The values increased from day 0 with a peak in biofilm development at 5 months, followed by a decrease in cell number to 102 CFU/coupon.
In general, the highest and lowest humidity values supported the best biofilm development as demonstrated by heterotrophic plate count data from the entire coupon surface. Intermediate RH values supported less biofilm growth, providing either stable values or decreased bacterial attachment over time.
When viewed by SEM, only small patches of biofilm formed on C22, N-316, and titanium coupon surfaces under conditions of less than 100% RH at 30°C (data not shown). Images from all time points and coupon types appeared similar to those depicted in Fig. 1A and C.
FIG. 1.
SEM imaging of biofilm development on titanium coupon surfaces at 100% RH, 30°C at day 0 (A), 5 months (B), and 12 months (C). Images for 12 and 18 months appeared the same for all metal coupon types.
Temperature effects on biofilm formation.
When the RH value was held at 100% and the temperature of incubation was held at 30°C, similar results were seen with all three metal types. There was an increase in values of HPC per coupon as early as 24 h of incubation, from approximately 102 CFU/coupon at day 0 to greater than 104 CFU/coupon. The values remained stable through 5 months of incubation, followed by a decrease in values for all metal coupon types by 18 months. With the C22 nickel alloy, a slight decrease in HPC values by 10-fold at 1 month was observed; however, the biofilm reestablished to the level of the other metal types by 5 months of incubation.
At 60 and 70°C, HPC values decreased for all three coupon types from less than 102 CFU per coupon to undetectable levels at day 1 and then remained undetectable for the duration of the test, with the exception of detectable values at 1 and 5 months of incubation in each case. The difference in HPC values at 60 and 70°C is significant compared to those at 30°C (Tables 4, 5, and 6).
TABLE 4.
HPC from N-316 stainless steel coupons over 18 months of incubation at 100% RH
| Incuba- tion period | HPC at temp (°C)a:
|
||
|---|---|---|---|
| 30 | 60 | 70 | |
| 0 days | 1.06 × 102 (2.7 × 101) | 1.06 × 102 (2.7 × 101) | 1.06 × 102 (2.7 × 101) |
| 1 day | 1.88 × 104 (2.18 × 103) | 0 | 0 |
| 1 mo | 3.88 × 104 (5.91 × 103) | 0 | 0 |
| 5 mo | 4.25 × 104 (3.30 × 103) | 78 (78) | 17 (11) |
| 12 mo | 8.78 × 103 (1.89 × 103) | 0 | 2 (2) |
| 18 mo | 7.61 × 102 (1.57 × 102) | 0 | 0 |
Standard errors were calculated from six replicates and are given in parentheses. All values were calculated from triplicate coupons.
TABLE 5.
HPC from C22 nickel alloy coupons over 18 months of incubation at 100% RH
| Incubation period | HPC at temp (°C)a:
|
||
|---|---|---|---|
| 30 | 60 | 70 | |
| 0 days | 53 (12) | 53 (12) | 53 (12) |
| 1 day | 2.98 × 104 (1.80 × 103) | 0 | 0 |
| 1 mo | 3.22 × 103 (8.30 × 102) | 0 | 3 (3) |
| 5 mo | 6.73 × 104 (1.23 × 104) | 44 (25) | 3 (2) |
| 12 mo | 4.88 × 103 (1.47 × 103) | 2 (2) | 0 |
| 18 mo | 5.04 × 102 (1.00 × 102) | 21 (21) | 9 (7) |
Standard errors were calculated from six replicates and are given in parentheses. All values were calculated from triplicate coupons.
TABLE 6.
HPC from titanium coupons over 18 months of incubation at 100% RH
| Incubation period | HPC at temp (°C)a:
|
||
|---|---|---|---|
| 30 | 60 | 70 | |
| 0 days | 56 (12) | 56 (12) | 56 (12) |
| 1 day | 3.65 × 104 (4.51 × 103) | 0 | 0 |
| 1 mo | 5.54 × 104 (1.31 × 104) | 36 (34) | 0 |
| 5 mo | 4.86 × 104 (1.90 × 104) | 12 (4) | 1.44 × 102 (1.39 × 102) |
| 12 mo | 1.22 × 104 (1.37 × 103) | 0 | 0 |
| 18 mo | 7.56 × 102 (8.60 × 101) | 0 | 0 |
Standard error values were calculated from six replicates and are given in parentheses. All values were calculated from triplicate coupons.
Biofilm formation was observed on the surfaces of the three metal coupon types under conditions of 100% RH and 30°C by using SEM. The titanium coupons (Fig. 1A to C) were indicative of biofilm development at day 0 and at 5 and 12 months on all three coupon types; in general, the coupons supported increased biofilm development through 5 months. By 18 months of incubation, SEM corroborated that a loss of biofilm had occurred (Fig. 1C). The images were supported by HPC values at the same time points.
At 60 or 70°C and 100% RH, biofilm development was negligible compared to that at 30°C, as evidenced by SEM on all three coupon types (data not shown).
Viable and culturable bacteria have been found in most subterranean environments (1, 4, 10, 13, 19, 21). Typical culturable counts range from 102 to 106 bacteria per cubic centimeter (19). Subsurface bacteria can be found alive and active, although their metabolisms proceed at slower rates than in surface environments (19). Subsurface environments have demonstrated high proportions of heterotrophic bacteria (4), especially in studies of oxygenated volcanic tuff at the Nevada Test Site (1, 10, 11).
When native microorganisms from the subsurface at Yucca Mountain were tested, temperatures from 60 to 70°C at 100% RH inhibited biofilm formation, resulting in few culturable counts above the level of detection. An exception was seen at the 5-month time point, where some biofilm attachment to the metal coupons was seen. Microorganisms capable of growth at 60 to 70°C would need to be thermophilic, with optimal growth temperatures of greater than 40°C (2). Even though the numbers and types of microorganisms present in the crushed rock were likely increased due to human and mechanical perturbation (12), they did not appear to include a robust population of thermophilic bacteria, as evidenced by the lack of biofilm establishment at elevated temperatures during the 18-month period of incubation.
In the present study biofilm formation might have been limited not only by elevated temperature but also by individual microbial ability to produce extracellular polymeric substances (EPS). Temperature may have had an effect on the production of EPS, which is known to enhance the adherence capability of bacterial cells. SEM studies have shown that Listeria monocytogenes cells produced an EPS matrix at 21°C but not at 10 or 35°C (17). EPS is critical not only for initial adhesion but in the firm anchorage of bacteria to solid surfaces (17) and in the ability of biofilm microorganisms to deal with environmental stresses, such as nutrient limitations, solar radiation, and variations in temperature (20). In this study there was negligible biofilm development imaged by SEM for all time points with elevated temperatures. General bacterial growth also was not observed on the coupons under the same conditions, implying that native bacterial cells were incapable of growth as well as biofilm production.
Results of both imaging and microbial HPC values demonstrated that 100% RH and 30°C were the optimal conditions for bacterial attachment and biofilm formation. HPC values at day 0 were approximately 1 × 102 CFU/coupon, and at 5 months of incubation the counts ranged from 4 × 104 to 7 × 104 CFU per coupon. At decreased RH values, ranging from 32 to 84% and 30°C, culturable counts were below or near the level of detection for all three metal types. Optimal conditions for aerobic soil microorganisms include a water activity value of 0.98 to 0.99 (2). However, some microorganisms can exhibit a greater tolerance for desiccation than others (24). Such desiccation tolerance was not exhibited in this study, as shown by the HPC values at 32, 70.5, and 84% RH compared to those at 100% RH (Tables 1, 2, and 3). Therefore, increased temperature to 60 or 70°C or decreased RH values below 100% impeded biofilm formation on metal surfaces used in this study.
Under optimal conditions within this study (100% RH and 30°C), HPC values from C22 nickel alloy coupons decreased from approximately 3 × 104 to 2 × 103 CFU between 1 day and 1 month. A possible explanation for this occurrence is that nickel is a heavy metal that is known to be toxic to bacterial cells (2, 6). However, as the biofilm developed the succeeding bacterial communities appeared to better tolerate the C22 nickel alloy. This was suggested by increased culturable counts from 2 × 103 CFU at 1 month to approximately 6 × 104 CFU at 5 months of incubation.
Under unsaturated conditions of soil and vadose rock, extensive biofilm development has not been observed (3). Biofilm development in this study followed the typical pattern of an unsaturated environment, namely, patchy development which was not extensive at any time during the 18 months.
Culturable bacterial counts from C22, N-316, and titanium coupons decreased by approximately 102 CFU per coupon between 5 and 18 months of incubation under optimal conditions. From 5 to 12 months, an exhaustion of nutrients and an increase in toxic waste by-products may account for the decreased CFU. Each metal surface resulted in a similar pattern.
No corrosion was observed on the metal coupon surfaces with SEM. Thiobacilli and sulfate-reducing bacteria, capable of metal corrosion, are known to exist in the rock and soil of the Nevada Test Site (5, 16, 21). Studies by Castro (5) have demonstrated metal coupon corrosion with Yucca Mountain rock and isolated cultures. Thiobacillus spp. and other iron-oxidizing bacteria are prevalent in the subsurface at the Nevada Test Site, but anaerobes, such as sulfate-reducing bacteria, can be found in anaerobic microenvironments within the subsurface (13, 21). For example, sulfate-reducing bacteria are the most abundant class of heterotrophs in the subsurface layers of the Savannah River Plant site (13). Perhaps the minimal biofilm development seen in this study or the length of the incubation time did not allow for visible corrosion effects. Visible corrosion has been observed on low-carbon steel metal surfaces under 100% RH conditions from barrels used to bury low-level radioactive wastes at the Nevada Test Site during the same timeframe as the present study (18).
This study demonstrates that muckpile rock excavated from a subterranean environment harbors microorganisms that can colonize metal surfaces, given the appropriate conditions. The boundary conditions observed in this study required 100% RH and 30°C for even minimal biofilm production. Other factors, such as metal type, nutrient availability, and toxicity, may have played a role in biofilm development as well. In the high-level nuclear repository environment of Yucca Mountain, initial high temperatures and low water activity are predicted to slow or eliminate biofilm formation. Over time, temperatures are predicted to decrease and water activity is predicted to increase during repository operation. These conditions will favor biofilm formation by surviving, indigenous microorganisms if nutrients are present for growth. Some indigenous bacteria within Yucca Mountain have been shown to be responsible for MIC (5), and others have been shown to survive, or resuscitate, after irradiation (22, 23). Therefore, the bounding limits of temperature and RH for biofilm formation will be important for predictive models of the stability of the high-level nuclear waste packages and support structures in the proposed repository.
Acknowledgments
This work was supported by the Department of Energy, Yucca Mountain Project, DE-FC08-98NV12081.
We thank Charles Neuwohner and Juhlpong Vilai for their technical assistance and the EPMA/SEM Facility, Department of Geosciences, University of Nevada, Las Vegas, for the use of the scanning electron microscope (Jeol JSM-5600).
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