Bacteriological characterization of wastewater samples obtained from a primary treatment system on a small scale swine farm

Bioresource Technology 101 (2010) 2938–2944

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Bacteriological characterization of wastewater samples obtained from a primary treatment system on a small scale swine farm A. Cordero, M. García, M. Herradora *, G. Ramírez, R. Martínez Department of Animal Production: Pigs, Faculty of Veterinary Medicine, National Autonomous University of Mexico, Av. Universidad # 3000, Mexico D.F., Mexico

a r t i c l e

i n f o

Article history: Received 18 October 2005 Received in revised form 5 December 2008 Accepted 6 December 2008 Available online 6 January 2010 Keywords: Swine farm residues Enteric bacteria Salmonella spp. Clostridium spp.

a b s t r a c t This study was carried out in order to quantify enteric bacteria and identify the presence of Salmonella spp., Escherichia coli, Clostridium perfringens and Erysipelothix rhusiopathiae in the liquid fraction of excreta generated from a small scale swine farm by means of a primary treatment system, consisting of the separation of solids and the sedimentation of liquids. Samples were collected at the following stages of the treatment: collection basin (CB), liquid obtained from a solids separator (SLF) and liquid from the sedimentation basin (SB). In each sample, enteric bacteria (cfu/g wet weight) and E. coli were quantified, Salmonella spp. was isolated and typified, and C. perfringens, and E. rhusiopathiae were isolated. No significant differences (p > 0.05) were found in the enteric bacteria and E. coli population levels at any treatment stage. S. choleraesuis was found in 20% of CB samples analyzed, 40% of SLF samples and 30% of SB samples. C. perfringens was isolated from SLF and SB. E. rhusiopathiae was not isolated (below the minimum detection limit). Results suggest that primary treatment does not reduce the amount of enteric bacteria, or eliminate Salmonella spp., E. coli and C. perfringens. Therefore, it is necessary to apply additional treatments to allow safe use of the liquid obtained in the farm. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Three production systems in Mexico may be classified in terms of their technological levels: (1) a full-technological system which represents approximately 55% of domestic production; (2) a partial-technological system that represents about 15%; and (3) a non-technological system accounting for the remaining 30% (Tinoco, 2004). The first two are used in swine breeding where high animal concentrations exist; this system is characterized by its high levels of noise, odors and amount of organic waste, feces and urine (León, 1995). Under this condition, the use of water for the removal of swine feces inside the farms can be as high as 50 L/pig/day, thus leading to the pollution of this non-renewable resource. Consequently, swine production farms are substantial pollution sources, particularly in those areas throughout the country with a high swine density (Pérez, 1997). A nationwide study found that 30% of the farms surveyed used residual water for crop irrigation and 38% disposed of it in lakes or rivers without treatment, while the remaining 32% discharged wastewater to public drainage (Pérez, 1997). Most larger (500 sows or more) farms (76%) have some sort of treatment, usually one that separates

* Corresponding author. Present address: Av. Rio de Guadalupe 42, San Juan de Aragón, México, Distrito Federal, CP 07950, Mexico. Fax: +52 55 56225870. E-mail addresses: [email protected], [email protected] (M. Herradora). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.11.072

solids and sends liquids to lagoons, but only 3% recycle water onsite (Pérez, 1997). Most of these treatments are practiced on a large scale, therefore, extensive equipment, space, large financial investment and increased of technology are required. Small producers, those with 100 sows or less, have no access to such technology. However, while the amount of wastewater does not vary substantially from medium (28–48 m3/day) to large farms (41 m3/day), there is a large difference when large farms are compared with small farms (24–80 m3/day), where a 50% more residual water per animal production unit is generated (Pérez, 2006). In small farms, remainder wastewater do not undergo any treatment or at most, only a primary treatment where excreta is first collected in a basin, solids are separated through a mechanical process and the liquid fraction is deposited in a sedimentation containers. This type of treatment increases wastewater biochemical oxygen demand (BOD), chemical demand (COD), total suspended solids (TSS) and total solids (TS). Nevertheless, treatment has little effect upon the bacterial load and the type of microorganisms that may remain in the liquids discharged from the farm into the environment, which becomes a potential health risk for both humans and other species (Taiganides et al., 1996). A variety of bacteria are able to survive and multiply in the pig manure during storage and disposal, including Salmonella spp., Escherichia coli, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Staphylococcus aureus, Clostridium perfringens, Bacillus anthracis,

A. Cordero et al. / Bioresource Technology 101 (2010) 2938–2944

Brucella spp., Leptospira spp. and Brachyspira hyodysenteriae (Strauch and Ballarini, 1994; Olson, 1995). Bacteriological analysis during previous studies has documented the presence of enteric bacteria aerobic and anaerobic samples (Iñigo et al., 1991; Mateu et al., 1992; Walker et al., 2008). It has been reported that enteric bacteria such as Salmonella spp. can survive for 13 months in swine manure (Gray and Fedorka-Cray, 2001). Salmonella spp. remains in the liquids contained in sedimentation basins at farms with different technological levels, sizes and environments (Ramírez et al., 2005). Pathogens such as C. perfringens and E. rhusiopathiae are able to survive in swine waste residues and infect other pigs exposed to the excreta. Since they are present in the liquids that the farm discharges, they could reach other animal populations and cause health problems in the environment (Madigan et al., 2000). It has been previously reported that species from the genus Clostridium resist environmental changes inside the sedimentation basin because they have the ability to sporulate (Hernández, 1997). An example is that Clostridium spp. concentration of 7.0  105 and 6.9  104 cfu/g of feces have been found, both in pig excreta processed in anaerobic systems with catch basins and in aerobic systems with flush tanks (Iñigo et al., 1991). E. rhusiopathiae can also be present in the liquids discharged from farms and may represent a risk to other swine operations (e.g. zoonotic infections) in nearby populations (Strauch and Ballarini, 1994). By evaluating the presence of bacteria such Salmonella spp., C. perfringens and E. rhusiopathiae in the excreta liquid fraction obtained from a primary treatment system at a small scale farm model, this study may determine whether it is necessary to make specific modifications in treatment systems to allow the elimination of pathogens from the liquids disposed of by the farm and then recycle them without risk of transmitting diseases. This information is important because many pig farmers use a primary treatment where the liquid fraction is directly spread onto vegetable crops or discharged into lakes and rivers, without any knowledge of the pathogen load and type it contains and the risk of transmission into other animal and human populations.

2939

basin that is 1.65 m wide, 1.95 m long and 2.45 m deep, and after two days, the liquid from the sedimentation basin (SB) generated is released to the local environment. The process takes seven days from when the material is poured into the CB until the SB is released. For purposes of objectives of this study, the entire system was emptied and restarted every week (Fig. 1). 2.3. Sampling CB, SLF and SB sample collections were performed once a week, for five weeks between April and May. Duplicate samples were collected every week for a total of 30 samples (2  5 CB, 2  5 SLF and 2  5 SB). Each sample consisted of five different subsamples of 200 ml, taken from five different areas within each sampling area (corners and center), and mixed to adjust to 1000 ml. Samples were collected by means of a water pump and a plastic pipes (Seconsa, México City, Mexico, Cat. 2011114) previously sterilized (in an autoclave in the laboratory), which were changed between CB, SLF and SB sample collection (Fig. 2). The 1000 ml samples (N = 30), were deposited in sterile glass containers (Quiromed, México City, Cat. 56); pH was measured using reactive strips (El Crisol, Mexico City, Cat. Hydr. 1423) and the temperature of the collected liquid was measured. The glass containers were labeled and placed in a polyurethane box with frozen packs (Polimex México City, Mexico, Cat. 015). The samples were kept at 4 °C during the two hour transport time to the laboratory.

2. Methods 2.1. Geographical location This experiment took place in a swine farm at Mexico State which is 2250 m above sea level, with 14.8 °C annual average temperature, and a 573.3 mm annual rainfall average in the wet season (July through September) (INEGI, 2004). The farm produces weaned pigs and it is designed for an average population of 120 breeding sows. The farm reported having had a 0.9% prevalence of erysipelas cases (4 sows), 1.14% of diarrhea caused by Salmonella spp. (5 sows) and 0.38% by C. perfringens (18 piglets) over two years. All cases were confirmed by bacteriological analysis in certified diagnosis laboratory (Department of Animal Production: Swine, diagnosis laboratory, Certificate RSGC 246). 2.2. Excreta treatment system The established swine excreta treatment system used in this farm is primary type and consists of three stages: a collection basin (CB) 2.34 m wide, 3.06 m long and 5 m deep, where the feces and wastewaters from every area of the farm are collected for storage. A pump for semisolids then sends this material to a cylindrical solids separator that yields a separated liquid fraction (SLF) and solids. The SLF subsequently goes to an anaerobic sedimentation

Fig. 1. Primary treatment system for residual water from a small scale swine farm, consisting of three stages: (1) collection basin, (2) solid and liquid separator, and (3) sedimentation basin.

2940

A. Cordero et al. / Bioresource Technology 101 (2010) 2938–2944

Influent (raw waste) I N

T h r o u g h

Solids

Effluent (treated waste)

O U T

CB (collection basin) SLF (solid/liquid fractionatior SBL (sedimentation basin) Sampling points Flow through the system

Fig. 2. Sampling points at the primary treatment system.

2.4. Bacteriological analysis 2.4.1. Enteric bacteria count In order to enumerate enteric bacteria, a tenfold serial dilution was carried out placing 1.0 ml of the sample into a 9 ml sterile saline solution. Fifty microlitre from each dilution were inoculated onto MacConkey Agar (MC) (Becton Dickinson of Mexico, Cuautitlan, Mexico, Cat. 220300) and incubated at 37 °C for 24 h, after which the number of colony forming units per gram wet weight (cfu/g) were counted and recorded (Martínez-Gamba et al., 2001). 2.4.2. Isolation and identification of Salmonella spp. To isolate and identify Salmonella spp., 10 ml were taken from each CB, SLF and SBL samples and inoculated into 90 ml of Sodium

Selenite Broth (BBL of Mexico, Izcalli, Mexico, Cat. 222641), as well as into Tetrathionate Broth (Becton Dickinson of Mexico, Cuautitlan Mexico, Cat. 211708), and incubated at 37 °C for 18–24 h. After this period of time they were re-inoculated via sterile swab procedure into Salmonella–Shigella Agar (SS) (Becton Dickinson, Cat. 214400) and Brilliant Green Agar (BG) (Becton Dickinson, Cat. 211708). A second passage was carried out on SS and BG 24 h later. After a third passage, the presence or absence of Salmonella spp. was confirmed (Krieg and Holt, 1984). Colonies with Salmonella spp. characteristics (transparent colonies with a central black point in the case of SS, and pink colonies in the case of BG) were selected and a smear was prepared from each colony. Those that corresponded to Gram-negative bacilli were transferred to plates containing SS and BG agar for

2941

A. Cordero et al. / Bioresource Technology 101 (2010) 2938–2944

presumptive confirmation. Twenty-four hours after the transfer, biochemical tests were run for identification by inoculating them onto Triple Sugar Iron Agar (TSI) (BBL, Cat. 252515), SIM-medium (Merck Sharp and Dome of Mexico, Mexico City, Mexico, Cat. 5470), Simmons Citrate Agar (BBL, Cat. 252514), urea (BBL, Cat. 252575), and sugars such as malonate, rhamnose, arabinose and threalose, and incubating for 24 h at 37 °C for final confirmation (Henry et al., 1995). Fifty microlitre of polyvalent antiserum of Salmonella somatic group O (Antiserum Poly A-I&Vi, Difco, Mexico City, Mexico, Cat. 222641) were placed in a glass plate, and a sterile bacteriologic loop was used to remove colonies from the TSI Agar that had previously been inoculated with Salmonella spp. A homogenized mix using the same bacteriologic loop was made in the next three minutes, while agglutination was assured as being representative of a positive reaction for additional confirmation. Once Salmonella spp. had been identified, 50 ll of the following antiserum were placed in a next glass plate: one from group C of the Salmonella somatic group O, group C1, factors 6 and 7 (BBL, Mexico, Cat. 222641); and one from group D1, factors 1, 9, and 12 (BBL, Mexico, Cat. 2951470). This test was done in order to differentiate S. choleraesuis from S. enterica. 2.4.3. Isolation of E. coli E. coli was isolated from the same CB, SLF and SB samples by inoculating 1.0 ml onto MC and incubated at 37 °C for 24 h. After that, colonies with apparent E. coli characteristics (pink, large and mucous) were selected, and a smear was prepared from each colony. Those that corresponded to Gram-negative bacilli and ‘‘coccobacilli” morphology were transferred to plates containing MC. Twenty-four hours later, a biochemical test consisting of TSI, SIM-medium, Simmons Citrate Agar and urea, was performed. After a 24 h incubation period, readings and records were made following a bacterial identification chart (Carter, 1994). 2.4.4. Identification of C. perfringens Fifty microlitre were taken from each original sample and inoculated into blood agar (Becton Dickinson, Cat. 211778) which had been previously incubated in two separate replicates for 48 h. One sample was incubated at 37 °C for 48 h under anaerobic conditions in polycarbonate jars (BBL, Cat. 260626), where CO2 was generated with hydrogen–carbon dioxide packets (Difco, Cat. 1952248). The second replicate was incubated in aerobic conditions as to determine whether the identified colonies were strictly anaerobic (Krieg and Holt, 1984; Carter, 1994). Colonies that appeared circular and grayish with a 1–3 mm diameter, smooth or with irregular borders and a double hemolysis were considered to suggest the presence of C. perfringens; and were selected and purified. Once a pure culture had been obtained, the colonies were Gram stained to confirm the presence of Gram positive bacilli. Positive colonies were Maneval stained in order to identify the capsule. Finally, the next biochemical tests were run to identify the colonies: Indole, Nitrate reduced, Gelatin, H2S, Urease, Meat (digestion), Milk and the carbohydrates arabinose, galactose, inositol, glucose, lactose, maltose, manitol, ramnose, raffinose, sacarose, sorbitol and trehalose (Quinn and Carter, 1994). 2.4.5. Identification of E. rhusiopathiae One millilitre from the sample was placed into 9 ml of 1% of buffered peptone water and incubated for 48 h at room temperature. Next, 50 ll were inoculated onto blood agar and incubated at 37 °C for another 48 h in a microaerophilic jar to providing an anaerobic condition by mean of the consumption of O2 with the use of a candle inside the jar. Characteristic colonies of 0.5–1 mm of diameter, smooth, convex, circular, transparent, with regular or irregular borders and an alpha hemolysis, were considered to

suggest the presence of the agent. These were Gram stained, and those that were Gram positive and filamentous bacilli were purified and examined through the next biochemical tests to confirm identification: Catalase, Voges-Proskauer, Nitrates reduced, Indole, Gelatin liquefaction, starch hydrolysis and the carbohydrates lactose, maltose, manitol, raffinose, salicin, sorbitol, sucrose, trehalose and xylose (Quinn and Carter, 1994). All bacteriological analyses were performed in the laboratory of the Swine Production Department in the Faculty of Veterinary Medicine at the National Autonomous University of Mexico, at Mexico City. 2.5. Statistical analysis Data were analyzed using a completely randomized design with three treatments (CB, SB and SLF). In order to analyze enteric bacteria counts (cfu/g) data were log transformed to base 10. Transformed data were used for an analysis of variance (ANOVA) with independent samples, applying Tukey’s test to compare means using the Statistical Analysis System package (SAS, Ver. 6.0, 1998). Salmonella spp. isolation data (presence or absence of the bacteria), were presented in a frequency model. 3. Results 3.1. Temperature, pH measures, enteric bacterial counts and E. coli isolation Temperature and pH for each stage of the system and the sampling are shown in Table 1. The greatest difference between the high and low pH and temperature measure were one pH unit or one degree celsius. No significant difference (p > 0.05) was found in the enteric bacteria counts carried out at every stage of the residual water treatment (Table 2). All samples were E. coli positive (6.4  105 – 5.5  106 cfu/g). However, no significant difference (p > 0.05) was found at any stage of the treatment system (Table 3). 3.2. Isolation and typification of Salmonella spp. Isolation of Salmonella spp. was achieved in 9 of 30 examined samples (30%) at different stages of treatment (Table 4). All of the positive samples corresponded to S. choleraesuis. 3.3. Isolation and typification of C. perfringens and E. rhusiopathiae C. perfringens positive results were obtained from two isolates (6.6%), one in from a SLF sample from sampling 1, and the other from the SB from sampling 5. Clostridium fallax and Clostridium oroticum were identified in SB from samplings 1 and 3. E. rhusiopathiae were below the minimal detection limit (MDL = 1 cfu/50 ll) in all samples collected from the three stages of the treatment system.

Table 1 Measurement of pH and temperature (°C) by week of sampling and stage of the system. Week

1 2 3 4 5 Mean a b c

CBa

SLFb

SBc

pH

T

pH

T

pH

T

8.5 7.5 7.5 8.0 7.5 7.8

18.0 18.0 17.0 18.0 17.0 17.6

8.5 7.5 7.5 8.5 7.5 7.9

18.0 18.0 17.0 18.0 17.0 17.6

7.5 7.5 7.5 8.5 8.0 7.8

18.0 18.0 17.0 18.0 17.0 17.6

Collection basin. Solid/liquid fractionators. Sedimentation basin.

2942

A. Cordero et al. / Bioresource Technology 101 (2010) 2938–2944

Table 2 Mean and standard deviations (SD) of enteric bacteria (cfu/g) by stage of the treatment system. Stage

Nd

cfu/ge

SDf

CBa SLFb SBc

10 10 10

2.6  106 2.1  107 9.0  106

1.5  106 6.2  107 2.4  107

Statistic differences were not found (p > 0.05). a Collection basin. b Liquid obtained from a solids separator. c Liquid from the sedimentation basin. d Sample size. e Mean colony forming units per gram of sample (wet weight). f Standard deviation.

Table 3 Average and standard deviations of E coli cfu/g (wet weight) by stage of treatment. Nd

Stage a

CB SLFb SBc

cfu/ge

SDf 5

10 10 10

7.4  105 8.6  105 1.8  107

6.4  10 6.7  105 5.5  106

Statistic differences were not found (p > 0.05). a Collection basin. b Liquid obtained from a solids separator. c Liquid from the sedimentation basin. d Sample size. e Mean colony forming units per gram of sample (wet weight). f SD = standard deviation.

Table 4 Number and percentage of positive samples to Salmonella spp. (S), Clostridium perfringens (Cp) and Erysipelothrix rhusiopathiae (Er) for each stage of treatment and sample week. Stage

CBa SLFb SBc Total a b c d e f

Nd

10 10 10 30

S

Cp

Er

ne

%f

ne

%f

ne

%f

2 4 3 9

20 40 30 30

0 1 1 2

0 10 10 6.66

0 0 0 0

0 0 0 0

Collection basin. Liquid obtained from a solids separator. Liquid from the sedimentation basin. Sample size. Positive sample. Percent positive sample.

4. Discussion 4.1. Temperature and pH findings Similar pH data between the stages of the water treatment were found in this study. These results do not agree with those reported by Strauch and Ballarini (1994), who found a pH decrease of 7.5– 6.5 in pig waste treatment residues, caused by production of fatty acids during the first month of storage; which later increased to 7. In the current study, pig excreta in the treatment system were stored for seven days and the pH range was from 7.5 to 8.5; which may be explained by the size of the farm (weaning pig production), less use of cleaning water, and the weekly re-starting system (that avoided the solids sedimentation). This may explain the lack of dilution of solids entering into the system. The temperature of the samples was steady for the different samples tested, with an average variation of a single centigrade degree. The ambient temperature during the sampling process was stable, so did not influence the temperature of the collected liquid.

4.2. Bacteriological enumerations The mean enteric bacteria counts (cfu/g) generated during this study from CB, SLF and SB were different from those found by other studies. Martínez-Gamba et al. (2001) reported values of 1.6  105 in the collection basin and 8  104 in the sedimentation basin, and Ramírez et al. (2005) reported values of 1.5  105 and 3.9  105 for similar cases. Both sets of data are lower concentrations than those obtained in the present study even though the authors took samples from farms with a significantly higher swine population that included fattening pigs units. In these farms the amount of liquid integrated into the wastewater system, was greater because the use of pigs refreshment system consist in damping pools inside the pens; therefore, there is a higher dilution of the bacterial load. While in this study the farm population was formed by a smaller number of breeding animals and suckling pigs not over four weeks old and there is not any hydraulic feces management. In the order hand, the shorter retention time of the system in the present study of seven days, did not allow enteric bacteria enough time to decrease. This can be explained by the study results of Mateu et al. (1992), in which samples taken from the sedimentation basin with material retained for 10, 14 and 18 days, found a 2.11  109 cfu/g concentration that decreased by day 18 to 2.87  107 cfu/g. For Salmonella spp. isolation, the percentage of positive samples in the present study (30%) was similar to that reported by Rajic et al. (2002), who isolated the agent in 31.76% of the 85 samples of drainage pipe material (25 g/ml). When comparing the CB positive samples with the SLF and SB samples, results do not concur with those reported by Hoszowski and Wasyl (2001), who identified a 54.5% of positive samples for Salmonella spp. from the effluent, but only 21.9% and 20.8% from the sediment and the compost. However Ramírez et al. (2005) reported the isolation of the bacteria in 70% of the collection basin samples, but only in 35% of the samples from the separated liquid and 30% from the settled liquid, respectively. The risk of transmitting Salmonella spp. through liquid wastes was proven by Jones (1976), who reported the isolation of Salmonella spp. in 76% of samples obtained from a liquid fraction stored for 30 days, and rendered a positive result even though there were no positively cultured animals at the time of the sampling. However, Baloda et al. (2001) isolated Salmonella in 90% of samples of sprinkler water from the liquid fraction. The importance of treating the liquid fraction from wastewater is obvious because some serotypes are able to survive in the environment for some (WHO, 2000; Cortinas, 2003). Henry et al. (1995) isolated 11 different Salmonella spp. serotypes from a facultative lagoon and from the effluent of three different farms. In the present study, when the typing of Salmonella spp. was done, it was observed that 100% was S. choleraesuis. This result does not concur with several other authors who have reported different Salmonella spp. serotypes, especially S. typhimurium. However, they do agree on the fact that serotype variety depends on geographical, environmental and immunity conditions (Baggesen et al., 1996; Davies et al., 2002; Rajic et al., 2002). With regards to the S. choleraesuis serotype, there is no information on percentages that indicates the presence of this agent in pig waste effluents to compare with other studies. However, Martínez-Gamba et al. (2001) noted the presence of S. choleraesuis in the liquid fraction of swine excreta following the same process in the three treatment stages and 7 day storage. These results indicate that the treatment system has no effect on the Salmonella spp. ability to remain viable in this manure treatment system. The E. coli count results in the present study were higher than those reported by Ramírez et al. (2005) at 5.3  104 cfu/g; and Mateu et al. (1992) at 1  104 cfu/g. However, they were similar to

A. Cordero et al. / Bioresource Technology 101 (2010) 2938–2944

those obtained by Iñigo et al. (1991), who studied fecal coliform contamination in fecal residues generated an aerobic process (1.1  105). These authors examined systems with a shorter retention time, which could account for the larger amount recovered in the present study. This is different from results of a study carried out by Jiang et al. (2002), in which they demonstrated that E. coli decreased from 0.5 to 2.0 log10 cfu/g in excreta after seven days at 15 °C. The presence of E. coli in swine wastewater is thoroughly documented, and its ability to survive for a long time in excreta has been demonstrated: 42–49 days at 37 °C, 49–56 days at 22 °C and 63–70 days at 5 °C (Ethan et al., 2002). Several authors have reported that enterotoxigenic and enterohemorrhagic E. coli have survived up to one year in fermentation lagoons (Van der Wolf and Peperkamp, 2001; Chern et al., 2004; Walker et al., 2008). The isolation of C. perfringens in the liquid fraction of swine excreta from the SLF and SB in spite of the treatment system agrees with the report by Iñigo et al. (1991) who identified several Clostridia spp., both in the swine excreta processed through anaerobic systems at catch basins and in aerobic systems. Additionally, Leung and Topp (2001) isolated Clostridium butyricum and Clostridium diporicum from the liquid fraction of swine excreta. C. perfringens sporulates to allow it to resist adverse environmental changes and survive in swine excreta for several months (Madigan et al., 2000). In the present study, C. perfringes was not identified in the collection basin, but was isolated in unprocessed residual liquid and in solid excreta. This may cause risk when residual water is employed in agriculture or is discharged into the environment or to sewage system and a higher risk if it is reused (Madigan et al., 2000). The characteristics of C. perfringens make harder it isolation, particularly because of the conditions of the waste materials from the farms, and because these agents can only be isolated in manure when their concentration is high, in contrast to enteric bacteria, which can be easily isolated by conventional cultures methods, even when their concentration is low (Strauch and Ballarini, 1994). Isolation of C. perfringens in material from the separator and the settled liquid indicates that mechanical separation and sedimentation is not totally efficient in the elimination of Clostridia spp. This is consistent with a report where C. perfringens could only be removed in 51% of sample tested at a large scale residual water treatment plant in Canada (Jones and Watkins, 1985). E. rhusiopathiae was below MDL from any of the samples at the present study. In contrast, some studies were able to isolate E. rhusiopathiae from different liquid manure sources. For example, the presence of E. rhusiopathiae and Clostridium spp. as pathogens in industrial waters has been reported by Strauch (1991). Although E. rhusiopathiae is a facultative anaerobic bacterium that can survive for 20 days or more in the ground and for six months in swine excreta, it does not have a capsule or produce spores. Therefore, it is important to take into account that the conditions the feces are exposed to in the stall or in the drainage system, the contact with disinfectants, exposure to drying conditions, and time spent in the collection basin may alter the time of bacterial survival.

5. Conclusions Conclusions drawn from the present study can be summarized as follows: (1) a swine manure wastewater primary treatment system, where the liquid fraction is obtained through a separation of solids and a sedimentation process, does not appear to reduce the enteric bacteria and E. coli load, (2) the identification of Salmonella spp. during this study indicates that it is not eliminated by a primary treatment process and therefore, remains a potential health

2943

risk. (3) The presence of C. perfringens in the liquid wastes from the farm indicates that primary treatment does not eliminate it; and consequently, this agent may be transmitted through the environment. These conclusions emphasize the need to identify systems to eliminate pathogens in the residual liquids of small scale farms in order to be able to recycle water while reducing risks of recycling pathogens into the farms. Acknowledgements This work was supported by the National Autonomous University of Mexico. DGAPA PAPIIT Project No. IN223903. References Baggesen, D.L., Wegener, H.C., Bager, F., Stege, H., Christensen, J., 1996. Herd prevalence of Salmonella enterica infections in Danish slaughter pigs determined by microbiological testing. Prev. Vet. Med. 26, 201–213. Baloda, B.S., Christensen, L., Trajcevska, S., 2001. Persistence of a Salmonella enterica serovar Typhimurium DT12 clone in a piggery and in agricultural soil amended with Salmonella-contaminated slurry. Appl. Environ. Microbiol. 67 (6), 2859– 2862. Carter, G.R., 1994. Bacteriología y Micología Veterinarias, 2da ed. El Manual Moderno, Mexico, D.F. Chern, C.E., Tsai, Y.L., Olson, H.B., 2004. Occurrence of genes associated with enterotoxigenic and enterohemorrhagic Escherichia coli in agricultural waste lagoons. Appl. Environ. Microbiol. 70, 356–362. Cortinas, N.P., 2003. Danger for health by water contamination. First International Workshop on Aquifer Vulnerability and Risk, May 29–30, Guanajuato, Mexico. National Water Commission, Mexico, p. 65. Davies, P., Funk, J., Turkson, P., O´Carroll, J., Nichols, M., Gebreyes, W., 2002. Effects of methods on the isolation of Salmonellas from preweaning piglets with diarrhoea in Vietnam. In: 17th Congress of the International Pig Veterinary Society, vol. 2, 2–5 June, Iowa State University, Ames, Iowa, USA, p. 42. Ethan, B.S., Sima, Y., Karl, R.M., 2002. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl. Environ. Microbiol. 1, 397–400. Gray, J.T., Fedorka-Cray, P.J., 2001. Survival and infectivity of Salmonella choleraesuis in swine feces. J. Food Prot. 64, 945–949. Henry, D.P., Frost, A.J., O’Boyle, D.A., Cameron, R.D., 1995. The isolation of Salmonellas from piggery waste water after orthodox pondage treatment. Aus. Vet. J. 72 (12), 478–479. Hernández, C.B., 1997. Determinación de bacterias patógenas en ensilados de excretas porcinas con caña de azúcar (Bachelor thesis). Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México. Hoszowski, A., Wasyl, D., 2001. Salmonella spp. found in waste, sewage sludge, compost and their antimicrobial resistance. Bull. Vet. Inst. Pulawy. 45, 163–170. INEGI (Instituto Nacional de Geografía e Informática), 2004. Anuario Estadístico del Estado de México. INEGI, Mexico, D.F., p. 120. Iñigo, D.C., Angelo, I.S., Soto, C.S., Alcaíno, H., 1991. Caracterización bacteriológica y parasitológica del desecho fecal porcino en Chile. Avances en Ciencias Veterinarias 6 (1), 23–28. Jiang, X., Morgan, J., Doyle, P.M., 2002. Fate de Escherichia coli O147:H7 in manureamended soil. Appl. Environ. Microbiol. 68 (5), 2605–2609. Jones, W.P., 1976. The effect of temperature, solids content and pH on the survival of Salmonellas in cattle slurry. Br. Vet. J. 132, 284. Jones, F., Watkins, J., 1985. Microbial aspects of water management. J. Appl. Bact., Symp. Suppl. 59, 27–36. Krieg, N.R., Holt, J.G., 1984. Bergey´s Manual of Systematic Bacteriology, vol. 2. Williams and Wilkins, Baltimore, USA. León, D.J.S., 1995. Impacto ecológico de la producción animal intensiva. El caso de la porcicultura. En: Luis Kato Maldonado (Ed.), La Producción Porcícola en México: Contribución al Desarrollo de Una Visión Integral. Universidad Autónoma Metropolitana, Mexico, pp. 277–290. Leung, K., Topp, E., 2001. Bacterial community dynamics in liquid swine manure during storage: molecular analysis using DGGE/PCR of 16S rDNA. FEMS Microbiol. Ecol. 38, 169–177. Madigan, M., Martinko, J., Parker, J., 2000. Brock Biología de los Microorganismos, 10th ed. Prentice Hall Madrid, España, p. 200. Martínez-Gamba, R., Pradal-Roa, P., Castrejón, P.F., Herradora, M., Galván, E., Mercado, C., 2001. Persistence of Escherichia coli, Salmonella choleraesuis, Aujeszky’s disease virus, Blue Eye disease virus in ensilages based on the solid fraction of the faeces. J. Appl. Microbiol. 91, 750–758. Mateu, A., Mata-Alvarez, J., Parés, R., 1992. Enterobacterial and viral decay experimental models for anaerobic digestion of piggery waste. Appl. Microbiol. Biotechnol. 38, 291–296. Olson, L.D., 1995. Survival of Serpulina hyodysenteriae in a effluent lagoon. J. Am. Vet. Med. Assoc. 207, 1470–1472. Pérez, E.R., 1997. Porcicultura y medio ambiente. Memorias II Seminario de Manejo y Reciclaje de Residuos Porcinos. Consejo Mexicano de Porcicultores, Octubre 22–25. Querétaro, México, pp. 10–12.

2944

A. Cordero et al. / Bioresource Technology 101 (2010) 2938–2944

Pérez, E.R., 2006. Granjas Porcinas y Medio Ambiente. Instituto de Investigaciones Económicas, Universidad Nacional Autónoma de México, México, D.F., p. 201. Quinn, P.J., Carter, G.R., 1994. Clinical Veterinary Microbiology, first ed. London, UK, p. 258. Rajic, A., Muckle, A., McFall, M., Deckert, A., Dewey, C., McEwen, S., Manninen, K., 2002. Salmonella occurrence and serotype diversity on 90 finishing farms in Alberta. In: 17th Congress of the International Pig Veterinary Society, vol. 1, 2–5 June, Iowa State University, Ames, Iowa, USA, p. 315. Ramírez, G., Martínez, R., Herradora, M., Castrejón, F., Galván, E., 2005. Isolation of Salmonella spp. from liquid and solid excreta prior to and following ensilage in ten swine farms located in central México. Bioresour. Technol. 96, 587–595. SAS. SAS/STAT., 1998. User Guide. Procedure Guide for Personal Computers. Ver. 6. SAS Institute Inc., Cary, NC, USA. Strauch, D., 1991. Survival of pathogenic microorganisms and parasites in excreta, manures and sewage sludge. In: Office International des Epizooties (Ed.),

Animals Pathogens and the Environment, vol. 10. Scientific and Technical Review, USA, p. 3. Strauch, D., Ballarini, G., 1994. Hygienic aspects of the production and agricultural use of animal wastes. J. Vet. Med. B 41, 176–228. Taiganides, P.E., Pérez, E.R., Girón, S.E., 1996. Manual Para el Manejo y Control de Aguas Residuales y Excretas Porcinas en México. Consejo Mexicano de Porcicultores, Octubre 22–25. Querétaro, México, pp. 77–80. Tinoco, J.L., 2004. La Porcicultura Mexicana y el TLCAN, primera ed. Universidad Nacional Autónoma de México, Mexico, p. 218. Van der Wolf, P.J., Peperkamp, N.H.T.M., 2001. Salmonellae types and their resistance patterns in pig faecal and post-mortem samples. Vet. Quart. 23, 175–181. Walker, P.M., Kelley, T.R., Smiciklas, K.D., 2008. Evaluation of pulverized trammel fines for use as a soil amendment. Bioresour. Technol. 99, 7848–7858. WHO, 2000. Chemistry Security International Program, second ed. World Health Organization.