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Applied and Environmental Microbiology, March 2006, p. 2287-2289, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2287-2289.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Recovery of Escherichia coli from Soil after Addition of Sterile Organic Wastes

Adrian Unc,^* Julie Gardner, and Susan Springthorpe

University of Ottawa, Centre for Research on Environmental Microbiology, Ottawa, Ontario, Canada

Received 17 June 2005/ Accepted 9 January 2006

Top Abstract Introduction References

 
Laboratory batch tests indicate that addition of sterile municipal sewage biosolids to clay soil from four depths increases the numbers of Escherichia coli isolates recoverable in EC-MUG broth (EC broth with 4-methylumbelliferyl-ß-glucuronide). This effect was most marked for the deeper soil layers, with increases of about 2.6 orders of magnitude in E. coli most probable number.

Top Abstract Introduction References

 
Widespread application of municipal biosolids to agricultural soils can result in direct, if largely temporary, contamination. Agricultural tile drains may transport pathogens and indicator bacteria from land applied organic wastes to water bodies. Recovery of Escherichia coli from tile drain discharges is considered to indicate potential pathogen transport and thus direct contamination from the surface applied biosolids; changes in number are commonly explained through filtration coefficients and die-off rates (4). Yet, it is known that E. coli bacteria from various sources may persist in soil over multiple years (7), independent of supplementary addition of wastes containing them. Over time, such contaminating E. coli isolates may become poorly cultivable or may not be cultivable at all on the routinely used glucuronide-containing media. It is also known that under appropriate nutrient conditions E. coli bacteria in water may increase in number (8). However, little is known about the potential for applied residuals to provide appropriate indirect nutrition to microbial contaminants remaining in soils from previous agricultural or other sources. Can changes in nutritional status in soil lead to improved regrowth and/or recovery of E. coli naturally occurring in soil, independent of the presence of a supplementary source of contaminant E. coli? If this is true, then observed increases in recoverable E. coli indicator bacteria may not necessarily be dependent solely on the addition of supplementary bacteria with land applied organic wastes.

We hypothesized that addition of biosolid nutrients to soil will enhance recovery of E. coli naturally found in soil.

Clay soil was carefully collected from four depths from the research station of Alfred College in eastern Ontario, Canada; characteristics of the soil from each of these four layers are given in Table 1. No organic wastes or fertilizers had been added to the soil for at least the last 10 years, during which time the land was fallow. The soil is not close to any water body, and we are not aware of any notable concentration of wildlife. The soil layers were air dried separately overnight, which was sufficient to allow sieving. Though it was recognized that the soil samples contained native bacteria, care was taken not to contaminate the samples further in the laboratory. Half of the soil from each layer was sterilized by repeated daily autoclaving over 3 days, and sterilization was verified by incubation of soil extracts on R2A agar and tryptic soy agar.

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TABLE 1. Properties of the clay soil layers used in the incubation tests

Treated, dewatered sewage sludge biosolids were collected from a wastewater treatment facility owned by a nearby municipality. Half of the biosolids retained for the tests were sterilized in a manner similar to that for the soil samples. Sterility of biosolid extracts was verified by use of tryptic soy agar and R2A agar as described above. The sterile biosolids were stored at 4°C, in the biohazard bags in which they were autoclaved, for 10 to 14 days before use.

Two grams of dry matter biosolids was added to 8 g of soil dry matter in sterile, 50-ml, loosely capped vials and incubated for up to 20 days at room temperature. It is expected that the conditions were mostly aerobic or microaerophilic during incubation. Water content was maintained at the field capacity level, which for this soil was calculated to be 51% (5), by addition of sterile deionized water. The tests included positive controls of fresh soil only (S_F) and fresh biosolids only (B_F), negative controls of sterile soil only (S_S) and sterile biosolids only (B_S), and mixed treatments of fresh soil and sterile biosolids (S_FB_S), sterile soil and sterile biosolids (S_SB_S), fresh soil and fresh biosolids (S_FB_F), and sterile soil and fresh biosolids (S_SB_F). The S_FB_S treatment was set up to answer the question posed in the hypothesis. Four vials from each treatment were destructively sampled at 0, 1, 2, 4, 7, 10, and 20 days. The content of each vial was added to 95 ml buffered saline solution (0.8% NaCl) (9) and shaken at 4°C with an Eberbach Corp. shaker at the high setting for 60 min. The resultant dilution was used to directly inoculate 96-well plates, or it was further diluted and then plated. Each well received 100 µl of sample on top of 100 µl of double-strength EC-MUG broth (EC broth with methylumbelliferyl-ß-glucuronide) (Difco Laboratories). The plates were incubated at 37.5 ± 0.5°C, and wells fluorescent under a long-wave UV lamp (366 nm) were counted after 24 and 48 h. Thus, a most probable number (MPN) was obtained for each 96-well plate (3). Presence of E. coli in the fluorescing wells was further verified by use of EC-BCIG agar (EC agar with 5-bromo-6-chloro-3-indolyl-ß-D-glucuronide) streak plates incubated at 37.5 ± 0.5°C for up to 5 days. About 10% of the fluorescing wells were verified this way for false-positive results; a similar proportion of nonfluorescing wells was verified for false-negative results. Statistical significances of differences among treatments and days were evaluated with standard t tests at a 0.05 significance level by use of the Minitab program (version 14.2; Minitab, Inc., State College, Pa.).

The results of the relevant incubations are shown in Fig. 1. After addition of fresh biosolids to fresh soils, the E. coli MPN rose immediately (Fig. 1), from just under 10² MPN g^–1 dry matter to about 10⁶ MPN g^–1 dry matter over the ensuing 4 days, reaching values significantly greater (day 4, P of <0.01 for all four soil layers) than those obtained with the treatments where fresh biosolids were added to sterile soils (between 10² and 10³ MPN g^–1 dry matter) (Fig. 1). The latter treatment did not lead to significant changes (P of >0.05 for all soil layers) in the E. coli numbers over the 20 days of testing (Fig. 1). This suggests that the numbers of E. coli bacteria added with the biosolids are augmented in natural soils by unknown mechanisms that involve existing biota. It is not known whether the contribution of soil biota to indicator numbers is made through cross-feeding through existing biota or whether the effect of nutrients from the biosolids acts directly on the E. coli bacteria. By 20 days, the numbers of recoverable E. coli isolates started to decline. In both of these incubation series, similar data were obtained for all four soil layers.

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FIG. 1. Changes in the numbers of E. coli organisms in the clay soil-biosolid mixtures. Symbols: , SFBF; , SSBF; (thick line), SFBS. Bars show 95% confidence intervals of the means; each point represents the mean of four samples. No E. coli recovery was observed for any of the all-sterile treatments or the fresh-soil control tests over the 20 days of testing. The figure was created with Corel Draw.

When sterile biosolids were added to fresh soil, the E. coli MPN was initially very low (<1 MPN g^–1 soil dry matter). However, increases of up to 2.62 orders of magnitude (Fig. 1) were observed. About 70% of the samples positive on EC-MUG were confirmed as E. coli on EC-BIG agar plates. For the superficial soil layers, the initial strong increase in E. coli MPN was not sustained into the 20th day of testing. For the deeper soil layers, which may be expected to contain different types and numbers of indigenous microorganisms (2), the E. coli values increased more slowly but consistently until the end of the testing period, with no signs of decrease.

While it is acknowledged that autoclaving likely modified the bioavailability of the biosolid nutrients and thus may have accelerated biological activity of soil biota, the data indicate that addition of organic nutrients to soil may result in an increase in the numbers of E. coli bacteria independent of the addition of E. coli in the waste material. Hence, a false microbial contamination indicator signal may be credited to the application of even relatively sterile organic wastes. This observation is significant, considering that the soil used in the testing was not treated with waste material for at least the last 10 years. Nevertheless, it is clearly necessary to understand whether the responses to nutrient addition would vary among E. coli strains from various sources, as well as whether the soil type and site location are of significance. Does the same response occur for enteropathogenic and enterotoxigenic strains of E. coli? Would E. coli regrowth response be even greater for soils that receive consistently organic wastes (e.g., manure from intensive animal operations and septage) containing or not containing significant numbers of pathogens and indicator bacteria?

Limited sole-nutrient-source tests, in fresh-soil-only treatments, indicated that individual N (NH₄NO₃), P (NaH₂PO₄ · H₂O), or C (dextrose) had a small (1.5 orders of magnitude) effect on the number of E. coli bacteria over about 4 days; the effect was similar for all soil layers. More testing is required in order to understand the role of each element and the synergistic effect of the combination of elements. It is interesting to note that survival of E. coli in soils amended with chemical fertilizers appeared to be prolonged (1); organic and chemical fertilizers are frequently applied to the same soils in the same year. Another interesting question, if nutrition of E. coli occurs through cross-feeding from other soil biota, is whether the composition of the soil microbial community is of significance in prolonging E. coli survival and/or in enhancing its regrowth. Clearly, more studies are needed to address these questions.

 
* Corresponding author. Mailing address: University of Ottawa, Centre for Research on Environmental Microbiology, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada. Phone: (613) 562-5800, ext. 8568. Fax: (613) 562-5452. E-mail: aunc{at}uottawa.ca.

Top Abstract Introduction References

 

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9. Wollum, A. G. 1986. Cultural methods for soil microorganisms, p. 781-802. In A. Klute, G. S. Campbell, R. D. Jackson, M. M. Mortland, and D. R. Nielsen (ed.), Methods of soil analysis, 2nd ed., part 2. Chemical and microbiological properties. American Society of Agronomy, Inc., and Soil Science Society of America, Inc., Madison, Wis.

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Applied and Environmental Microbiology, March 2006, p. 2287-2289, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2287-2289.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Unc, A. Articles by Springthorpe, S. Search for Related Content PubMed PubMed Citation Articles by Unc, A. Articles by Springthorpe, S. Agricola Articles by Unc, A. Articles by Springthorpe, S.