Impact of calcium carbonate and temperature on survival of Escherichia coli in soil

Journal of Environmental Management 119 (2013) 13e19

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Impact of calcium carbonate and temperature on survival of Escherichia coli in soil M.B. Farhangi a, *, A.A. Safari Sinegani a, M.R. Mosaddeghi b, A. Unc c, G. Khodakaramian d a

Department of Soil Science, College of Agriculture, Bu-Ali Sina University, Hamadan 65174, Iran Department of Soil Science, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran c Plant and Environmental Sciences, New Mexico State University, Box 30003 MSC 3Q, Las Cruces, NM 88003-8003, USA d Department of Plant Protection, College of Agriculture, Bu-Ali Sina University, Hamadan 65174, Iran b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 August 2012 Received in revised form 3 January 2013 Accepted 27 January 2013 Available online 19 February 2013

Spreading of waste organic matter on agricultural lands is considered to enhance soil microbial activities and physical properties and improves soil nutrient status. However, organic wastes have also been shown to be a source of microbial contaminants including pathogens. Related risks are governed by pathogens’ survival and transport particularities. We evaluated the significance of high levels of CaCO3, common in arid and semi-arid soils, on survival of Escherichia coli NAR at different temperatures. Amendments of 0, 5, 10, 15 or 25 g CaCO3 were mixed into variable soil amounts to obtain 100 g soileCaCO3 mixtures. Both sterile and non-sterile soil mixtures were tested. Suspensions of a nalidixic acid-resistant E. coli strain (E. coli NAR) were added to the mixtures at a rate of 106 cell g
1 soil. Mixtures were incubated at 4, 15, or 37  C at the soil’s field capacity for water (i.e. 0.13 g g
1). Each treatment was tested in triplicate. Persistence of culturable E. coli NAR was verified throughout the incubation period. The recovery rates of culturable E. coli NAR were significantly correlated to CaCO3 concentrations (P < 0.05). Incubation temperature (T) was the most significant factor (P < 0.01). In non-sterile mixtures the largest decline in survival rates of E. coli NAR was measured for treatments with larger CaCO3 content (i.e. 15 and 25%). Interaction of temperature and CaCO3 was significant for E. coli NAR die-off. Sterilization of soil caused nonuniform fluctuations in the effect of treatments. The maximum calculated decay rate for E. coli NAR was 0.83 d
1 for the 15 g CaCO3 non-sterile mixture incubated at 37  C while the minimum was 0.09 d
1 for the control unamended sterile soil incubated at 15  C. A combination of high temperature, large CaCO3 concentrations and a non-sterile, biologically active soil created the least favorable conditions for E. coli survival. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: CaCO3 Temperature Escherichia coli NAR Survival Decay rate

1. Introduction Spreading of sewage sludge on agricultural lands has been gaining attention in past decade as it improves soil nutrient status and enhances microbial activity (Johansson et al., 1999). Sewage sludge has been shown to improve soil physical properties including porosity, structure, bulk density, aggregation, and especially water holding capacity (Logan et al., 1996). On the other hand pathogens including bacteria, viruses, protozoa, annelid and nematode helminths and fungi have been found in sewage sludge (Epstein, 1998). Although different sludge stabilization processes provide reduction of pathogen population, complete elimination is rarely (if ever) achieved (Ngole et al., 2006). In many arid regions in developing countries the main source of drinking water is

* Corresponding author. Tel.: þ98 811 4227013; fax: þ98 811 4227012. E-mail address: [email protected] (M.B. Farhangi). 0301-4797/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvman.2013.01.022

groundwater abstracted from dug or drilled wells (Foppen, 2002). Moreover, wastewater collection and treatment systems are uncommon in these countries and therefore there is a real risk for the abstracted water being contaminated with pathogens and pose a health risk through the food chain (Foppen, 2002). Percolation through the vadose zone has been found to be a significant mechanism for removing microbial contaminants (Gerba and Bitton, 1984). Several experiments have shown that microorganisms in septic tank effluent may be nearly completely removed after percolation through a relatively short distance in unsaturated porous media (Bouma et al., 1972; Ziebell et al., 1974). Jiang et al. (2005, 2006) noted that bacteria retention occurred mostly in the top layer of a sand column (top 0.1 m for their case study). The main retention mechanisms are physical straining or filtration within the pores of the solid matrix (Tan et al., 1992; Wan et al., 1994; Wan and Tokunaga, 1997; Schäfer et al., 1998; Mosaddeghi et al., 2009, 2010). Depending on its physiological state a bacterial cell may attach to surfaces where it may grow or can be

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dislodged into the aqueous solution and transported to new regions. Soils as filtration media for percolating wastes carrying bacterial contaminants may allow their survival and growth and thus serve as a temporary sink and a source for delayed leaching of bacteria into the subsurface (Ralfs, 2007). Several factors have been reported to affect the survival of fecal coliforms (FC) in soils. According to Gerba and Smith (2005), animal and human waste-borne pathogenic bacteria may survive in soil from 2 months to 1 year at common or optimum conditions, respectively. Safari Sinegani and Maghsoudi (2011) reported survival of E. coli to range from 40 days to 3 month in soils treated with manures, depending on the source of manure and soils water content. The 40 days survival interval was noted for soils treated with poultry manure or sewage sludge while in saturated soils treated with cow manure Escherichia coli survival extended to over 90 days. Kirby et al. (2003) also reported that soil moisture, temperature, and texture affected the survival of FC both in laboratory and in field studies. Survival of bacteria decreases with increasing temperature (e.g. Kristiansen, 1981; Stenstrøm and Hoffner, 1982). Unc and Goss (2006) found that survival of E. coli in soils amended with swine or cow manure decreased as temperature increased. van Donsel et al. (1967) reported 90% reduction of FC in the soil to occur within 13.4 days in winter but within only 3.3 days in summer. Foppen and Schijven (2006) reviewed extensively studies reporting on effects of temperature on E. coli die-off rates. Despite the heterogeneous conditions among the various experiments reviewed, the dependency of the die-off rate coefficients on temperature was similar; the increase in the die-off rate coefficient per degree Celsius rise was apparent and comparable in most experiments. Inactivation or die-off rate quantitatively relates microbial inactivation or dying-off in terms of log10 decline per day (John and Rose, 2005). Much research focused on E. coli O157:H7, the most dangerous serovar of E. coli known in cattle manure (Jones, 1999). While varying among soil types, survival of this serovar in soil might be extensive (Guan and Holley, 2003). In laboratory it survived for at least 56 days at 25  C (Mubiru et al., 2000), and under fluctuating environmental temperatures (
6.5 to 19.6  C), its presence was detected for up to 99 days (Bolton et al., 1999). The extended survival of fecal coliforms in organic soils compared with that in mineral soils has been proposed as possibly due to the higher water-holding capacity of organic soils (Gerba and Bitton, 1984). Semi-arid and arid region soils have low organic matter and are rich in carbonates with calcic or petrocalcic horizons commonly occurring. More than 50% of the latter can be CaCO3 (Ruellan, 2002). Cuthbert et al. (1950) showed that E. coli survive several weeks in limestone (pH ¼ 5.8e7.8) while dying in a few days in a peat soil (pH ¼ 2.9e4.5), indicating the importance of pH as well as water content. While there is no information on the survival of fecal microorganisms in CaCO3 rich soils we could find the work of Bashan et al. (1995) who studied the survival of Azospirillum brasilense in 23 types of plant-free sterilized soils obtained from a wide range of environments (containing 0.5e48.4% CaCO3) in Israel and Mexico. Counts of A. brasilense declined rapidly in 35 days after inoculation in Israeli soils (arid, semi-arid, or mountain regions), but remained stable or even increased during the 45-day incubation in the arid soils from Mexico. Bashan et al. (1995) therefore reported CaCO3 to be among the abiotic parameters that controlled survival of A. brasilense and that only at its largest concentrations CaCO3 had a highly negative effect on viability of A. brasiliense in soil. Although interactions of carbonates and bacteria have been studied for microbial-induced carbonate precipitation processes, in

an engineering context, the role of this mineral on survival of FC has not been specifically evaluated. To the best of authors’ knowledge, there is no published information on the combined effect of carbonate and temperature on survival of E. coli. The objective of our study was to elucidate the significance of calcium carbonate on survival of E. coli, a well-known indicator of FC, at different temperatures. Proper understanding the factors governing environmental survival of microbes and sludge-borne pathogens could offer an objective basis for defining appropriate management options. 2. Materials and methods 2.1. Soil collection and analysis In October 2010 soil was collected from the surface layer (0e 15 cm) of an agricultural site in Hamadan city, northwestern Iran. The collection area is in a semi-arid climate with annual precipitation of 328 mm, and annual average temperature of 13  C (maxima of 35.5  C recorded in August and a minima of
10.3  C recorded in January). The collected soil was air-dried and passed through a 2-mm sieve before further analyses. Soil particle-size was analyzed by the hydrometer method (Gee and Bauder, 1986). Equivalent calcium carbonate (ECC) was measured by the backtitration procedure (Loeppert and Suarez, 1996). Soil acidity (pH) and electrical conductivity (EC) were determined in a 1:2 soil:water extract (Hesse, 1971). Organic carbon content (OC) was determined by the wet-oxidation method (Walkley and Black, 1934). Heterotrophic bacterial and fungal populations in the soil were estimated by the plate count method. Soil samples were diluted in sterile 0.2% Na4P2O7 solution. Nutrient agar (NA), Rose Bengal starch casein nitrate agar (RBSCNA), and modified potato dextrose agar (MPDA) were used for determination of total soil bacteria, Actinomycetes or fungi. Counts of microorganisms were expressed as log10 colony forming units (CFU) per gram dry mass of soil sample (log10 CFU g
1). Basal respiration (BR) was measured as CO2 rate evolved over 5 days per gram of soil dry mass (Alef and Nannipieri, 1995). 2.2. SoileCaCO3 mixtures preparation The sampled soil was not calcareous with considerably low ECC (0.20 g 100 g
1). The soileCaCO3 mixtures were prepared by mixing 5, 10, 15 or 25 g analytical-grade CaCO3 (Merck, Darmstadt) into 95, 90, 85 or 75 g soil (dry mass basis) and poured into 150-mL polypropylene incubation vials. These CaCO3 concentrations are not common for lime amending scenarios, where usually 1.35e9.78 Mg lime is added to a hectare (varying by soil type) for a unit change in the soil pH (CPHA, 2002), but occur in subsurface calcic or petrocalcic horizons in arid land soils. Thus our experimental setup considers such extreme conditions as a means to establish potential mechanisms/effects relevant for waste infiltration into such soils. Unamended soil samples were used as control. Half of each mixture was sterilized over 5 d through a 2 h daily autoclaving at 121  C as per Unc and Goss (2006). Between each autoclaving, the mixtures were kept under aseptic conditions at room temperature to facilitate germination of spores. To compensate for any gravimetrically detected evaporative loss during sterilization processes sterile deionized water (less than 50 mL per 100 g mixture) was added to the mixtures. Heat sterilization allowed assessing the absolute impact of abiotic soil properties on E. coli NAR survival. The effectiveness of the autoclaving was evaluated by plating dilutions of autoclaved mixtures on Nutrient agar (NA). No growth was detected after 48 h for any mixture type.

M.B. Farhangi et al. / Journal of Environmental Management 119 (2013) 13e19

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2.3. Bacterial inoculums preparation

2.6. Data analysis

We used a nalidixic acid-resistant E. coli strain (E. coli NAR), kindly provided by the Pasteur Institute of Iran. This strain is nonpathogenic, rarely found in natural environments, and possesses survival characteristic similar to other E. coli strains (Jamieson et al., 2004). It occurs singly or in pairs and is classified as facultative anaerobic, chemo-organotrophic bacterium (Abu-Ashour et al., 1998). Inoculum was produced by transferring a loopful of E. coli NAR from a plate culture to a 250-mL Erlenmeyer flask containing 100 mL trypticasesoy broth (TSB) followed by incubation at 37  C for 14e16 h (Jamieson et al., 2004). Afterward, cells were harvested by centrifugation at 5000  g for 20 min. Cells were washed twice with sterile physiological saline (0.85% NaCl) and re-suspended in saline solution. The final suspension had approximately 1e 2  107 cells mL
1. This was determined via the absorbance at 600 nm (OD600 ¼ 1 by Varian, Cari 100 UVevis spectrophotometer) and verified by cell counting on a Neubauer Improved Bright-Line Hemacytometer (Prolab Scientific).

Statistical tests were carried out on logarithmically transformed (base 10) CFU counts (log10 CFU g
1). A value of 1 was added to the raw counts before transformation to avoid the loss of the undefined values at the detection limit (i.e. CFU of 0). Significance of factors for the survival of E. coli NAR was evaluated using a general linear model (GLM). For some sampling events the log10 CFU g
1 data was not normally distributed (ShapiroeWilk test) and thus the nonparametric KruskaleWallis model was employed. The role of CaCO3 concentrations and incubation temperatures has been evaluated separately for the non-sterile and sterile mixtures treatments. These analyses were carried out for each sampling event. To facilitate comparisons between the treatments, the decay rate constant (k) was estimated, by fitting experimental results to the following first-order decay equation (Eq. (1)):

2.4. Bacterial survival experimental design A multi-level factorial experiment with complete randomized design was employed, with soil sterility status (non-sterile and sterile), CaCO3 level (0, 5, 10, 15, 25 g per 100 g), and incubation temperature (4, 15, and 37  C) as factors. Temperature ranges are representative of environmental temperatures for the studied area at late fall and late winter (4  C), early spring (15  C), or summer (37  C). This combination of factors (2  5  3) produced 30 treatments. All incubations were performed in triplicate, in laboratory, under controlled conditions, in individual sterile looselytied polypropylene vials that allowed gas exchange. As water availability (i.e. soil water potential) can obscure the significance of other factors for bacterial survival (Unc and Goss, 2003), the gravimetric water content of the mixtures was adjusted to 0.13 g g
1. This corresponds approximately to the field capacity for water of the soil without CaCO3 (control) and was not significantly affected by CaCO3 addition. During incubation water was added every third day as necessary to maintain the soil water content at field capacity. 2.5. Sampling and enumeration of E. coli NAR Samples were obtained from the initial mixes and during incubation at 0.5, 1, 2, 3 and 8 d, then every 5 d for the first month, every 10 d during the second month, and eventually every 15 d afterward. At each sampling three vials from each treatment were collected and destructively tested for the presence and counts of E. coli NAR. For extraction of the E. coli NAR a 1 g (dry mass) soil mixture sample was added to 99 mL Na4P2O7 0.2% sterile solution and mechanically shaken for 20 min at 200 rpm (Wollum, 1986). Subsequent decimal dilutions were prepared as necessary to produce 30 to 300 colonies per plate from a 0.1 mL dilution aliquot. Recovery plates were prepared with methylene blue (EMB) agar supplemented with 100 mg mL
1 nalidixic acid (Abu-Ashour et al., 1998). Plates were incubated at 37  C for 24 h. Average CFU counts were normalized per dry soil mixture mass (CFU g
1). Enumeration continued until no bacteria were detected on the plate. The detection limit of the spread plate method was 100 CFU g
1 soil (Nyberg et al., 2010), but sampling continued after reaching the detection limit to verify the possibility of bacterial re-growth.

Nt ¼ N0  exp
kt

(1)

where Nt is bacterial CFU g
1 at time t and N0 is bacterial CFU g
1 at time t ¼ 0. The fitted decay rate constants were compared between treatments by Student’s t test. Data analysis and graphing were carried out on SAS 9.12 (SAS Institute, 1990) and MS-Excel. 3. Results and discussion Results illustrate the dynamics of E. coli NAR persistence at three temperatures in sterile and non-sterile soils amended with different levels of CaCO3 at constant water content close to field capacity. Thus water availability was not a factor. A laboratory-grown labeled bacterial strain, E. coli NAR, was selected, because of its easy detection. The use of heat sterilization allowed assessing the significance of abiotic soil properties for survival of E. coli NAR. 3.1. Physical, chemical and biological properties of the studied soil The soil was texturally a loam (50, 35 and 15 g 100 g
1 sand, silt and clay respectively) and had relatively high organic carbon content (1.30 g 100 g
1) for an arid land soil. Therefore it is likely that it did not impose organic matter limitations on heterotrophic microbial growth. The soil had an EC of 0.13 dS m
1(1:2 soil:water extract). Soil pH was in a moderate range (7.26) not deleterious to most bacteria including E. coli. Soil resident microbial counts for bacteria, fungi and Actinomycetes were 6.66, 3.98 and 4.55 log10 CFU g
1, respectively, indicating a biologically active soil. Soil basal respiration was 0.15 mg CO2 d
1 g
1. Microbial resident counts and basal respiration were determined at the field capacity for water (0.13 g g
1) and at 25  C. 3.2. Survival of E. coli NAR in CaCO3 amended soil In non-sterile mixtures, analyses of variance showed a significant effects (P < 0.01) for the proportion of CaCO3 on culturable E. coli NAR for the 0.5, 1, 2, 3, 33 and 53 d sampling. Temperature (T) and the CaCO3  T interaction were significant (mostly P < 0.01) at all sampling events in both sterile and non-sterile mixtures. The log10 counts of E. coli NAR were normally distributed (Shapiroe Wilk; P < 0.05) at all sampling times except for days 8 and 13 in non-sterile mixtures. Non-parametric statistical tests (Table 1) have shown the effects of CaCO3 and T to be significant also for days 8 and 13 (P < 0.01). For the sterilized mixtures counts of E. coli NAR often showed non-uniform decay patterns in contrast to those obtained from non-sterile mixtures (Figs. 1 and 2). However, for the sterilized

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Table 1 Analysis of variance (c2-value) of culturable E. coli NAR in soil as affected by CaCO3 and incubation temperature (T). Sampling event (day)

8 13 33 43 53 63 78

Chi-square parameter (c2) Non-sterile

Sterile

42.9681** 41.4363** e e e e e

43.1800** 42.5735** 43.4663** 43.7885** 43.7364** 43.8737** 43.7288**

Values marked by ** are significant at P < 0.01.

mixtures the effects of CaCO3 on culturable E. coli NAR data were significant (P < 0.05 and P < 0.01) at all sampling times except for 18 d; T and CaCO3  T interaction were also significant (P < 0.01) at all sampling times. In sterilized mixtures, log10-transformed counts

Fig. 2. Changes in counts of culturable E. coli NAR over time in sterile soileCaCO3 mixtures at 4  C (a), 15  C (b), and 37  C (c).

of E. coli NAR were normally distributed (ShapiroeWilk test; P < 0.05) till 28 d sampling time (except for 8 d and 13 d). Nonparametric statistics was used for all non-normally distributed datasets. Results for 8 d and 13 d and from 33 d to 78 d are presented in Table 1; all indicate a significant role for both CaCO3 and T (P < 0.01). 3.3. Decay of culturable E. coli NAR counts in non-sterile soile CaCO3 mixtures

Fig. 1. Changes in counts of culturable E. coli NAR over time in non-sterile soileCaCO3 mixtures at 4  C (a), 15  C (b), and 37  C (c).

Changes in the counts of culturable E. coli NAR in non-sterile mixtures are presented in Fig. 1. At 4  C there was a relatively accelerated die-off during the first two days (Fig. 1a) ranging between 1 and 3 orders of magnitude across the treatments followed by a slight recovery in culturability of 1e1.5 log CFU g
1. This occurred across all treatments including control and thus was likely a result of the initial inoculation shock, likely due to the shift in temperature. While culturability varied across treatments the two larger CaCO3 concentration (i.e. 15% and 25%) led to the most rapid count decay bringing the E. coli NAR reaching nondetection at 43 days of incubation. For all other treatments E. coli

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NAR counts reached the detection limit after 63 days of incubation. Counts in the 10% CaCO3 treatment, for reasons that we cannot infer from our measurements, fluctuated distinctly from all other treatments. Thus for the 4  C treatments, after excluding the 10% CaCO3, the minimum best fitted decay rate constant (Table 2) was allocated to the control treatment (0.15 d
1) whereas the 15% and 25% CaCO3 treatments had the maximum value (0.21 d
1). Corral et al. (1988) noted that E. coli could be killed by sodium bicarbonate treatment, but significant killing would occur as the pH became more alkaline (pH 8.6). At alkalinization the bicarbonate anion undergoes an additional dissociation and the carbonate anion concentration increases. In our study addition of CaCO3 increased soil pH by about 0.4e0.6 units. Therefore all mixtures could be classified as slightly alkaline (pH > 7.5). Diez-Gonzalez et al. (2000) indicated that carbonate and alkali treatment of cattle manure virtually killed all E. coli in only 5 days. Arthurs et al. (2001) concluded that carbonate anion rather than bicarbonate anion is the antibacterial agent and carbonate stoichiometry is a more important variable than time per se. Bicarbonate is a relatively nonreactive monovalent anion but carbonate anion forms insoluble complexes with divalent cations (Mgþ2, Caþ2, Znþ2, and Feþ2) that play an important role in bacterial physiology (White, 1995). At 15  C there was no large post-inoculation decrease in bacterial counts (Fig. 1b) confirming the role of the temperature shock in the colder treatment. For most of the incubation period the E. coli NAR counts were similar across treatments suggesting that factors other than CaCO3 mainly governed survival. Moderate temperatures (15  C) are suitable for rapid growth of psychrophilic and mesophilic soil bacteria and those able to tolerate CaCO3 might compete for carbon sources with E. coli in non-sterile soil. A direct comparison between the equivalent non-sterile and sterile treatments (Figs. 1b and 2b) suggests a role for the microorganisms native to soil. Protozoa may ingest E. coli (predation), while indigenous bacteria may compete for the same resources (Foppen and Schijven, 2006). Foppen and Schijven (2006) reported higher die-off rate coefficient for non-sterile conditions, where predation and/or antagonism may be of significance, as compared to sterile conditions. Arthrobacter spp. and pseudomonads, the organotrophic bacteria most commonly found in soil, might be some of the competitors. Streptomyces spp., the most commonly found (nearly 70%) Actinomycetes in arid and semi-arid soils, also grow well at pH ranging between 6.5 and 8; they are well-known for their capability to synthesize antibiotics, as secondary metabolites, harmful to other microorganisms (Giri et al., 2005). In our study, highest decay rate constants were calculated for non-sterile mixtures containing 0, 15, and 25% CaCO3 at 15  C (Table 2). For the non-sterile 15  C treatments non-detection occurred at the same time as for the 4  C treatments. Non-detection was reached on day 43 for the 15% and 25% CaCO3 and on day 63 for the other treatments. Expectedly, the decay constants were also rather

17

similarly grouped, largest for treatments with larger CaCO3 content (Table 2). Sjogren (1994) has shown more rapid E. coli die off at 10 and 20  C in an alkaline soil versus an acidic soil (1.5e2 times faster) but with little difference between the two soils at 5 or 37  C. In our case incubation at 37  C resulted in different trends. Two findings were obvious: 1) survival was drastically shortened for all treatments when compared to the 4 and 15  C treatments, and 2) the two larger concentrations of CaCO3 (15% and 25%) killed E. coli NAR exceedingly faster. The calculated decay rate constants for 15% and 25% CaCO3 were 0.83 and 0.78 d
1, respectively, about four times larger than those for the control treatment (0.23 d
1) (Table 2). Since E. coli commonly resides in the gastrointestinal tracts of warm-blooded animals it is expected to survive at similar environmental temperatures (i.e. 37  C). However, it seems that complexity of soil environment and presence of large CaCO3 levels undeniably affected E. coli NAR survival at 37  C. Unc and Goss (2004) reported that cool temperature favors bacterial survival in soil. Low temperature, as limiting factor, can slow down microbial metabolism and maintain bacteria viability until the limiting factor is removed (Bogosian et al., 1996). Semenov et al. (2007)reported a significant decline in survival of E. coli O157:H7 and Salmonella typhimurium as mean temperatures increased from 7 to 33  C. Ishii et al. (2006) found that naturalized E. coli strains survived longer when incubated at 4, 15, and 25  C than when incubated at 30  C or 37  C. 3.4. Decay of culturable E. coli NAR counts in sterile soileCaCO3 mixtures Fecal organisms introduced to thermally sterilized soils would be faced with a new environment which has some favorable parameters such as absence of microflora that could interfere or even suppress them, the presence of lysed cells’ residues that provide plentiful organic substrates and inorganic moieties essential for growth and reduced concentrations of inhibitory compounds which are unstable at the high temperatures of sterilization (Mitscherlich and Marth, 1984). Accordingly the net effects of abiotic parameters manipulation may be studied in isolation. Obviously, the main disadvantage of sterility is that it occurs almost never in nature and that any results obtained under sterile condition may be exaggerated. Counts of E. coli NAR during incubation at 4  C in sterilized soils are presented in Fig. 2a. The effects of soil sterilization on survival were considerable for all treatments (Fig. 1a). No E. coli NAR could be detected after less than one month of incubation at 4  C for any of the sterilized soileCaCO3 treatments. At this temperature the minimum decay rate constant (0.29 d
1) was calculated for the control treatment (Table 2). Results for incubation of sterile mixtures at 15  C are presented at Fig. 2b. After 12 h (0.5 d) of incubation E. coli NAR counts

Table 2 Fitted decay rate constants, k (days
1), for E. coli NAR. Temperature ( C)

Non-sterile 4 15 37 Sterile 4 15 37

CaCO3 level (%) 0

5

10

15

25

0.16 (0.85)** 0.16 (0.83)** 0.24 (0.88)**

0.17 (0.89)** 0.16 (0.85)** 0.24 (0.88)**

0.14 (0.73)** 0.15 (0.81)** 0.26 (0.89)**

0.21 (0.74)** 0.22 (0.83)** 0.83 (0.80)**

0.21 (0.76)** 0.20 (0.72)** 0.79 (0.72)*

0.29 (0.53)* 0.09 (0.60)** 0.21 (0.75)**

0.60 (0.80)** 0.15 (0.91)** 0.35 (0.76)**

0.35 (0.76)** 0.16 (0.91)** 0.44 (0.74)**

0.30 (0.60)** 0.10 (0.72)** 0.38 (0.55)*

0.56 (0.76)** 0.13 (0.80)** 0.24 (0.46)*

Values marked by * and ** are significant at P < 0.05 and P < 0.01, respectively. Values in parentheses denote R2 for the fitted log-linear regressions.

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increased slightly above the initial inoculation in all mixtures. This might be assigned as a benefit of soil sterilization as discussed previously. Certainly, the role of temperature on bacterial growth was important since this trend was not observed at 4 or 37  C. After about a month of relative stability of E. coli NAR counts a rapid decay was measured. It is important to note that starting with the third day of incubation bacterial counts in the control treatment were consistently the largest confirming a role for CaCO3. The longest survival (3 months) was measured for control and 15% CaCO3. Accordingly the control treatment had the lowest decay rate constant in this study (0.09, Table 2). After 78 days of incubation, no E. coli NAR could be detected in any CaCO3 amended treatments. Incubation of sterile mixtures at 37  C caused an immediate, sharp decrease in E. coli NAR counts in all CaCO3 amended mixtures (3.5e4 log units), a trend similar to the equivalent non-sterile treatments but more severe (Fig. 2c). Decay was most accentuated in the two treatments with the largest CaCO3 content. The control treatment was the least impacted. These results again confirmed the role of CaCO3 in inhibiting E. coli survival. While, for the 37  C incubation, sterilization of soil enhanced survival of E. coli in the CaCO3 amended treatments (see Figs. 1c and 2c) it had no effect for its survival in the control treatment at this incubation temperature. The later observation suggests the overriding significance that temperature has above the soil sterilization status. 3.5. Decay rate constants (k) The estimated decay rates (k) and regression coefficients (R2) for E .coli NAR die-off are presented in Table 2. In general, for the nonsterilized soils, larger decay rates were estimated for the 37  C treatments (Table 2). The differences, up to 4 times larger than those at 4  C and 15  C, were statistically significant (Student’s t test, P < 0.01). For the mixtures containing 15% and 25% CaCO3 decay rates were larger than for all other concentrations of CaCO3 including controls. Incubating sterilized mixtures containing 5% or 25% CaCO3 at 4  C led to larger average decay rates (Student’s t test, P < 0.01) compared to other two incubation temperatures. It must be remarked that the sterilized control, non-amended, treatments (0% CaCO3) had the smallest decay rates at all three incubation temperatures. However, the same was not as clearly defined for the non-sterilized soils. While the sterilized soil treatments were of use in discriminating the role of CaCO3 amendments in limiting E. coli survival the overall shortest survival occurred under the high temperature and large CaCO3 concentration in non-sterilized soils suggesting a complementary role for the soil’s biotic parameters. Craig et al. (2004) and Lee et al. (2006) demonstrated the positive effects of organic matter on E. coli survival and Bashan et al. (1995) reported the negative effect of CaCO3 on A. brasilense survival in soil. Bashan and Vazquez (2000), carried out tests in natural and artificial soils, and concluded that CaCO3 and sand negatively affected survival of Azopirillium spp. However, in the rhizosphere where the bacteria benefited from plentiful organic matter, survival period was prolonged and the role of other factors like CaCO3 alleviated. We confirmed that such an effect is likely also valid for E. coli. By ignoring experiments under sterile conditions and assuming that all other experimental variations are negligible, Foppen and Schijven (2006) concluded that for E. coli the average die-off coefficient at 10  C is 0.15 d
1 and at 20  C it is 0.50 d
1. In the absence of CaCO3, our results suggest possibly slightly lower decay coefficients but we must note that these were obtained from arid soils which are likely less biologically active. While the CaCO3 amendments in our study are surrogates for carbonated soils of arid and semi-arid regions, our results suggest that calcium carbonate rich arid soils may have the capability to minimize microbial risks

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