Water Air Soil Pollut (2009) 199:151–157 DOI 10.1007/s11270-008-9867-9
Factors that Influence the Transport of Bacillus cereus Spores through Sand Minyoung Kim & Stephanie A. Boone & Charles P. Gerba
Received: 26 June 2007 / Accepted: 13 September 2008 / Published online: 18 October 2008 # Springer Science + Business Media B.V. 2008
Abstract The goal of this study is to clarify the surface-chemical and microphysical variables that influence bacterial spore transport through soil, thereby defining the factors that may affect spore transport velocity. Bacillus cereus spores were continuously monitored in a soil column under saturated conditions with experimental variations in soil grain size (0.359 and 0.718 mm), pH (7.2 and 8.5), and water flow rate (1.3 and 3.0 mL/min). Increasing soil grain size, flow rate, and pH resulted in enhanced spore movement. Spore transport increased 82% when soil grain size was doubled. An increase in effluent flow rate from 1.3 to 3.0 mL/min increased spore movement by 71%. An increase in pH increased
M. Kim (*) National Institute of Agricultural Engineering, Rural Development Administration, 249 Seodun-dong, Gwonson-gu, Suwon 441–707, South Korea e-mail:
[email protected] S. A. Boone Southern Regional Research Center (SRRC), USDA-ARS, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, USA e-mail:
[email protected] C. P. Gerba Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, AZ 85721, USA e-mail:
[email protected]
spore transport by 53%. The increase in hydrodynamic forces resulting from the larger grain size soil and higher flow rate functioned to overcome the hydrophobic nature of the spore’s coat, and the interparticle bonding forces between the spore and soil particles. Keywords Groundwater contamination . Spore transport . Bacillus cereus . pH . Flow rate . Soil grain size
1 Introduction About 40% of the United States’ domestic water supply originates from groundwater, and over 40 million people obtain drinking water via individual wells from groundwater (Alley et al. 1999). In the U.S. between 1971 and 1994, at least 356 disease outbreaks have been attributed to contaminated groundwater, representing 58% of all waterborne illness outbreaks (Craun and Calderon 1997). Use of reclaimed water and improper wastewater disposal are cited as current reasons for groundwater contamination. In addition, the intentional release of pathogenic microorganisms associated with bioterrorism has highlighted the possibility of increases in disease and fatality due to groundwater contamination. Of particular concern is the spore of Bacillus anthracis, the causative agent of anthrax in humans and animals, which can survive for decades in the soil (Ricca et al. 2004). The bacteria
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spore’s ability to survive for long time periods under extreme conditions is one reason B. anthracis has been used by terrorists (Arakawa et al. 2003). Understanding the variables that influence microbial transport through environmental media is the first step toward assessing the feasibility, risk, and impact of distributing pathogenic microbes to target sites. Several studies have indicated that viral and bacterial transport through soil is influenced by many parameters, including the unique properties of microbes, solution chemistry, soil characteristics, and environmental physical properties such as interstitial fluid velocity (Bales et al. 1995; Gannon et al. 1991). Research indicates that water flow rate, pH, and soil grain size are the most significant factors influencing virus and bacteria transport (Taylor et al. 2004; Huysman and Verstraete 1993; Lindqvist and Bengtsson 1995). In contrast to the large number of studies investigating virus and bacterial transport, very few studies were conducted using bacterial spores. Sinton et al. (2000) used pore size exclusion to explain the transport retardation of rhodamine WT dye, followed by MS2, Bacillus subtilis spores, E. coli J6-2, and E. coli 2690. Schijven et al. (2003) found that MS2 and Clostridium bifermentans spores can travel greater distances than Escherichia coli (E. coli). Research by Pang et al. (2005) indicated that spores have a greater attachment to gravel when compared to viruses and bacteria. Overall, previously conducted studies have focused on spore transport by comparing their movement to other microbes, but the effect of environmental variation on spore transport has not been clarified. To date, there are no studies found that examined the surface-chemical or physical factors affecting bacterial spore transport. Therefore, this study is the first to investigate the effect of physical factors (soil grain size and water flow rate) and surface-chemical interaction of pH on the process of spore transport through soil.
2 Materials and Methods 2.1 Spore and Sporulation There is a greater than 99% sequence similarity in the primary structures of 16S rRNA in B. anthracis and B. cereus. In addition, both Bacillus species are found in soil, are pathogenic and Gram-positive, and form endospores which are highly resistant to heat,
Water Air Soil Pollut (2009) 199:151–157
chemicals, and radiation. The endospores of B. cereus and B. anthracis are similar in morphology as they both have thick hydrophobic spore coats, a cortex, exhibit pilli, no S-layer, a unique peptidoglycan core wall, and have calcium dipicolinic acid present in the core (Stalheim and Granum 2001; Anderson et al. 2005). Therefore, nonpathogenic B. cereus spores were used as a surrogate for B. anthracis. B. cereus species 129 T (BGSC #6A1) were obtained from the Bacillus Genetic Stocks Center in Columbus, Ohio. A vegetative cell suspension was prepared from the B. cereus strain 129 T after growth in sporulation media consisting of 8 g DIFCO nutrient broth, 1 g KCl, and 0.25 g MgSO4.7H2O per liter of deionized water supplemented with 1 mL of 1 M CaCl2, 1 mL of 0.01 M MnCl2, and 1 mL of 0.001 M FeSO4 (Schaeffer et al. 1965). Sporulation medium was autoclaved at 121°C for 1 h. After inoculation, the suspension was incubated at 37°C for 72 to 110 h to obtain the correct spore titer of 108. Prior to spore purification, light microscopy was used to verify the spore count. The sporulation suspension was placed in a 250-mL plastic bottle and centrifuged at 9,500 rpm for 10 min. The supernatant was poured off, and the pellet was washed with a quarter culture volume of 1 M KCl and 0.5 M of NaCl. Centrifugation was repeated, and the pellet was then resuspended in 25 mL of 50 mM Tris–HCl (pH 7.2) containing 1% (w/v) maltose. The resulting pellet was incubated at 37°C for 1 h and then washed in succession with 1 M NaCl, deionized water and 0.05% sodium dodecyl sulfate (SDS). The pellet was then washed three additional times in deionized water. Spores were heat-shocked at 80°C for 10 min and stored at 4°C until use (Schaeffer et al. 1965). 2.2 Soil Column Each experiment was carried out using commercially available Accusand (North Kato Supply LLC, Mankato, MN, USA) sieved through 20/30 mesh ðcoarse sand ¼ 0:718 0:123mmÞ and 40/50 mesh ðmedium sand ¼ 0:359 0:062mmÞ openings. The course sand has a bulk density of 1.83 g/cm3 and porosity of 0.35. The medium sand has a bulk density of 1.80 g/cm3 and porosity of 0.37. Generally, Accusand has a rounded shape, specific gravity of 2.65 g/cm3, a moisture content of
