Abstract

Elevated level of Salmonella in dairy farm generated wastewater can contaminate food and water. Controlling the risk of Salmonella infection requires improving the existing understanding of Salmonella decay in impaired dairy wastewater. Enhanced understanding of Salmonella inactivation in dairy wastewater can help deriving improved animal waste management practices capable of mitigating the risk of pathogen contamination to cropland as well as water resources. Considering the importance of the animal waste borne pathogen issue, the primary objective of the study was set to determine the degradation pattern of Salmonella at mesophilic environment (37 °C). To do so, the impact of sampling timing (morning vs evening) on the changes in Salmonella counts were assessed. Further, a heat stress study was conducted to identify the critical die-off time at thermophilic temperatures (48 and 58°C). Results from the study found that there was a 5.2 log10 reduction in Salmonella count observed over the thirteen days study period. There was no significant difference in Salmonella count during the sampling of morning or evening. Heat stress study showed that the first 30 minutes was the major die-off time. Regrowth of Salmonella was observed at thermophilic temperature after two days of further incubation. The outcome of the study will help to understand pathogen inactivation in dairy wastewater, and derive improved animal waste treatment methods.

1 Introduction

The liquid and solid manures produced in feedlots such as dairy farms are great sources of nutrients and often used as fertilizers to enhance soil nutrients, and thus crop yields. Annually, ~2.2 billion t cattle manure is generated in the United States, and potentially applied to croplands (USDA 2009; USDA 2014). While the application of animal manure to cropland reduces dependence on chemical fertilizer for replenishing soil nutrients, manure borne contamination such as pathogens also poses risks to water, soil and environmental health. Controlling the risk of contamination in vegetables and crops grown on land receiving manure as fertilizer (Tauxe 1997; Natvig et al. 2002; Erickson et al. 2014) and ambient water requires reducing pathogen and bacterial loads in manure.

In manure, the presence of many pathogens including E. coli O157:H7, Salmonella spp., Campylobacter, Clostridium perfringens and Listeria monocytogens are reported (Zhao et al. 1995; Pell 1997). Food borne pathogens such as Salmonella and E. coli are known to be present in the gastrointestinal tracts of ruminant animals (Laven et al. 2003; Wells et al. 2001), leading to the sporadic shedding of these pathogens in their feces (Erickson et al. 2014; Rhoades et al. 2009) which can cause manure borne pathogen contamination, and thus infections.

Previously linkages between agricultural soils receiving animal manure and the risk to the microbiological safety of fresh produce have been shown (Ongeng et al. 2011; Bach et al. 2002; Hutchison et al. 2004). Increasing numbers of outbreak incidents are reported to have association with fresh vegetables and food-borne infections (Doyle and Erickson 2008; Barak et al. 2005; Beuchat 2002). Further, applying animal manure to cropland can contribute to microbial pollution in ambient water (Pandey et al. 2014; Soupir et al. 2006). Unmanaged runoff from cropland which receives manure as fertilizer can carry pathogens, which can contaminate shellfish areas and cause many water quality problems (USEPA 2012).

The linkages between manure application, pathogen contamination in crops (Natvig et al. 2002; Islam et al. 2004a, 2004b) and ambient water (Pandey et al. 2012) indicate the need for additional pathogen control measures with regard to manure management and its application for agricultural purposes. One option for controlling the transport of pathogens to food crops and water is that all manure must be treated prior to application as fertilizer (Erickson et al. 2014). Untreated manure or the manure, which have not been stored for sufficient time intervals for inactivating pathogens, can facilitate transfer of pathogens to ready-to-eat crops (Islam et al. 2004a; Natvig et al. 2002; Toth et al. 2012), as well as to surface and ground water. Natvig et al. (2002) showed, using a controlled environment chamber study, that vegetables such as arugula and radishes could be contaminated by applying bovine manure inoculated with Salmonella serovar Typhimurium or fecal Escherichia coli; however, the level of contamination on vegetables could be reduced by managing the timing of manure application and crop harvest.

Applying untreated dairy manure immediately after cattle excretion has a greater potential to contaminate vegetables and crops grown in the cropland, so the use of appropriate methods, capable of inactivating pathogens, is needed to reduce the risk of contamination. Methods such as drying (Amin-Nayyeri et al. 2010), aerobic digestion (Dumas et al. 2010), anaerobic digestion (Aitken et al. 2005; Popat et al. 2010; Pandey and Soupir 2011), composting (Maeda et al. 2010; Erickson et al. 2014), heat treatment (Shepherd et al. 2010; Garcia et al. 2010) and radiation treatment (Sinton et al. 2007; Brandon et al. 1977) are proposed for treating the manure and controlling manure borne pathogens.

Many factors, including pH, temperature, mixing, non-mixing, feedstock, C:N (carbon:nitrogen) ratio, incubation time, aerobic condition and anaerobic environment, influence pathogen survival (Toth et al. 2012; Allievi et al. 1994; Pandey et al. 2015; Erickson et al. 2014; Kim et al. 2009; Harikishan and Sung 2003; Hipsey et al. 2008). However, one of the most important factors regulating the survival of pathogens is temperature, particularly when pH levels lie in the neutral range (pH = ~7.0) (John and Rose 2005; Hipsey et al. 2008).

In the dairy industry, excessive amounts of both liquid and solid manure (within the neutral pH range 6.5–8.0) are produced (Pandey et al. 2015). Figure 1 shows how this is typically handled.

Figure 1 Flow of manure in a dairy system.

A considerable amount of water is used for cleaning and flushing the barns which house the cow herd, and subsequently the flushed water is collected in storage ponds (i.e. lagoon systems) before being applied to the cropland. The solid portion is stored in the form of compost piles. The flushed water includes feces, urine and bedding materials, which are also the major reservoir of pathogens (Murinda et al. 2004; Hutchison et al. 2005; Toth et al. 2013). The recycling of dairy wastewater (which is likely to have elevated bacterial loads and is stored in ponds) for cleaning the barns is a normal practice which also poses the risk of recirculation of pathogens. Previous research reported that pathogens can survive in dairy wastewater for extended periods of time (Kearney et al. 1993; Wang et al. 1996; Heinonen-Tanski et al. 1998; Himathongkham et al. 1999; Himathongkham and Riemann 1999; You et al. 2006).

Controlling dairy wastewater borne pathogens is critical because of the health risks associated with food and water borne pathogens. To mitigate Salmonella infections in food and water, improvement in the existing understanding of Salmonella decay in contaminated liquid and solid manure is needed. Enhanced understanding of Salmonella inactivation in dairy wastewater can help in deriving suitable treatment methods capable of controlling animal waste borne pathogens. To improve the understanding of wastewater borne Salmonella inactivation, a laboratory scale benchtop study was designed, and the impacts of various thermal conditions on Salmonella inactivation in dairy wastewater were assessed. The specific objectives of the study were:

• to determine the degradation pattern of Salmonella count in a mesophilic environment (37 °C);
• to evaluate the impact of the sampling event (morning vs evening) on Salmonella count; and
• to evaluate die-off time during heat stress testing in a thermophilic environment (48 °C and 58 °C).

2 Material and Methods

2.1 Feedstock Preparation

Fresh dairy manure was collected from the on-campus dairy facility of the University of California, Davis. The fresh dairy manure was frozen at −20 °C and was thawed prior to the experiment. About 4.27 kg manure was thawed and mixed with 4.5 L of deionized water. The pH of the mixture was 7.27. The mixture was then sieved through a 850 µm (ASTM #20) mesh to separate the fibrous residue from the wastewater. The moisture content of the manure water was 98% with pH = 7.7.

2.2 Experiment Setup

A bench scale setup, shown in Figure 2, was designed using an overhead mixer (Carfamo Limited Model BDC 250), heating chamber and temperature sensor. Initially an inoculated wastewater sample was collected to enumerate the concentration of pathogens at the beginning of the experiment. The wastewater was placed inside a heating chamber to ensure constant heating at 37 °C. The wastewater was mixed continuously with the overhead mixture at 50 rpm. The inactivation of Salmonella typhimurium (ST) in wastewater was observed for 2 weeks. The samples of wastewater were collected twice (morning, 09:00, and evening, 16:00) during the first 3 d and once for the next 11 d incubation. For the heat stress study on d 10 of incubation, the incubated (10 d) wastewater samples were placed in two additional chambers running at thermophilic temperatures (48 °C and 58 °C) for 2 h. The samples were collected at 30 min intervals to observe the inactivation of ST in dairy wastewater at elevated temperatures.

Figure 2 Schematic of experimental setup.

2.3 Pathogen Inoculation, Enumeration and Statistical Analysis

Dairy wastewater was inoculated with the strain of Salmonella typhimurium LT2 (ATCC # 700720). The ST was grown overnight in Luria-Bertani broth (Difco LB Broth, Miller; Becton, Dickinson and Company, Sparks, Maryland) on a bench top incubator shaker (MaxQ 4000, Thermo Scientific, Ohio) at 100 rpm and 37 °C for 24 h. To ensure the quality control, a negative control of the respective growth media was used. The initial concentration of ST in inoculum was enumerated. Afterwards, 30 mL pure culture of ST was centrifuged at 10 000 rpm for 15 min to form the pellet. The pellet of ST was dissolved in 5 mL phosphate buffered saline (PBS), and then it was added and mixed (for 15 min) with the dairy wastewater.

To enumerate the ST, the dairy wastewater sample was serially diluted in PBS, platted (duplicate), and incubated for 24 h at 35 °C to ensure the ST growth before enumeration. ST enumeration was carried out following the Bacteriological Analytical Manual (BAM) protocol (USFDA 2014a). XLD (xylose lysine desoxycholate) agar (Becton, Dickinson and Company) was used for the isolation and differentiation of ST. The ST appears as red–yellow with black centre in the agar plates.

 The first statistical analysis was conducted to evaluate the change in ST concentration at mesophilic temperature (37 °C) over the 14 d experiment. The second statistical analysis of ST was conducted to evaluate the impact of sampling events between morning and evening at 37 °C for the first 3 d. The third analysis was performed to evaluate the change in ST concentration over time during the heat stress study at thermophilic temperatures. A comparative analysis of ST was carried out to evaluate the impact of sample timing on the heat stress study and among the three temperatures (37 °C, 48 °C and 58 °C) to evaluate the inactivation of ST in the late phase of the experiment (i.e. the last 4 d incubation) in mesophilic and thermophilic conditions. All the statistical analyses were performed using PROC GLIMMIX in SAS (Littell et al. 1996). An alpha level α = 0.05 was used to identify significant differences among treatments by least significant difference methods.

3 Results and Discussions

3.1 ST Inactivation in Dairy Wastewater at Mesophilic Environment

The concentration of ST in the inoculated dairy wastewater (at room temperature) was 1.6 × 10⁹ CFU/mL, shown in Figure 3. Black bars indicate Salmonella levels. Small letters in the top of bars show statistical differences in the Salmonella typhimurium levels. Similar (alphabetically adjacent) letters show no differences, and different letters show significant differences at α = 0.05.

Figure 3 Changes in the levels of Salmonella typhimurium LT2 (average with standard error) in manure water at 37 °C.

The ST levels in wastewater were reduced significantly (p < 0.000 1) over the 14 d incubation period at 37 °C. On the first day of incubation at 37 °C, the ST count reduced from 1.6 x 10⁹ CFU/mL to 4.6 × 10⁶ CFU/mL (2.5 log₁₀ CFU/mL reduction). The ST count then increased and remained stable around 1.5 × 10⁸ CFU/mL (8.2 log₁₀ CFU/mL) over the next 7 d. Subsequently, the ST counts were reduced steadily (4.3 log₁₀ CFU/mL) and reached the levels of 1.1 × 10⁴ CFU/mL on d 14. There was a 5.2 log₁₀ CFU/mL reduction in ST numbers over the course of the 14 d incubation period.

Several previous studies examined the inactivation pattern of Salmonella at different environmental and physiochemical conditions. You et al. (2006) studied the inactivation of a multidrug resistant Salmonella serovar Newport strain and a drug susceptible strain in dairy manure and found that the Salmonella count increased during the first 3 d incubation. Subsequently, the Salmonella levels decreased until 35 d incubation. A rapid drop in concentration to below detection level was observed by 49 d (184 d by the most probable number method) under ambient temperature conditions (24.5 °C ±1.4 °C) with changing moisture (weekly loss <3% total weight). Toth et al. (2011) found a comparable survival pattern of Salmonella where the increase in concentration was followed by a steady log-linear decline phase, and finally a long tailing phase of low Salmonella concentrations. The authors observed that Salmonella can survive >137 d (by the most probable number method) in the effluent of a dairy lagoon under the natural conditions of southeastern Pennsylvania. The log reduction time (days required for a 90% reduction of concentration) was reported to be 7 d.

The survival pattern of ST in the current study differs from the patterns of Salmonella reductions reported by Toth et al (2011) and You et al. (2006), which can be attributed to the differences in dairy wastewater. Additionally, the current study was executed in a controlled condition reducing the risk of an ambient environment. The decline in ST concentration was not log linear over the incubation period. A study by Kearney et al. (1993) found that the decline in Salmonella count in cattle slurry was temperature dependent during a 135 d storage period. They found that Salmonella levels declined faster at an elevated temperature (17 °C vs 4 °C). The period required to achieve a 1 log₁₀ unit reduction was 21.3 d at 4 °C compared to 17.5 d at 17 °C. Another study by Himathongkham et al. (1999) found a long reduction time of 12.7 d for Salmonella in dairy manure slurry at 20 °C. The pH of dairy wastewater varied within the range 7.6 to 8.0 during the time of experiment. You et al. (2006) found that the increase in pH from 6.7 (at 0 d) to 7.6 (at 37 d) and 8 (at 72 d) was linked with the decrease in Salmonella count. Other studies, such as Himathongkham et al. (1999) and Wang et al. (1996), also reported possible relationship between the increases in pH with the decline in pathogen concentration.

The dry matter content in the dairy wastewater was 3% to 4% for this experiment. Hutchison et al (2005) found that 7 d and 9 d of incubation (correspondingly) was required to incur a 1 log reduction in concentration when Salmonella spp. inoculated dairy slurries containing high (7.2%) and low (2.1%) amounts of dry matter were exposed at atmospheric temperature in storage tanks. The authors predicted that the phenomenon of drying, rather than other parameters such as dry matter, ould be a crucial influence on the survival of Salmonella in dairy slurry. Nicholson et al (2005) found a maximum Salmonella survival of 32 d in dairy slurry with 7% dry matter compared to 93 d survival in dairy slurry with 2% dry matter at temperature ~20 °C. However, a laboratory scale study by Jones (1976) found a longer survival time of Salmonella in cattle slurry containing 5% dry matter as compared to 1%.

3.2 Diurnal Effect on ST Inactivation in Mesophilic Temperature

The ST levels in morning and evening samples are shown in Figure 4, and indicate the change in ST levels within a day. Black bars indicate Salmonella levels in samples collected in the evening. Hollow bars indicate Salmonella levels in samples collected in the morning. Small letters in the top of bars show statistical differences in the Salmonella typhimurium levels. Similar (alphabetically adjacent)letters show no differences, and different letters show significant differences at α = 0.05.

Figure 4 Changes in concentration of Salmonella typhimurium (average with standard error) in manure water at 37 °C during the first 3 d of the experiment.

While analyzing the morning and evening samples, there was no significant interaction between day and time of the sampling period (p = 0.062). However, there was significant difference in the total count of ST at different days (p < 0.002). The count of ST was low in d 1 compared to d 2 and d 3 (Figure 4). Thus, the average count of ST may not change over a short span of time of ≤8 h, but it may change over a longer duration such as ≥24 h, which emphasizes that ST inactivation may not occur drastically in a mesophilic temperature at 37 °C in dairy wastewater.

3.3 Impact of Heat Stress Post Treatment on ST Inactivation

The reduction in ST levels during of heat stress test (on d 10) is shown in Figure 5 below. Black bars indicate Salmonella levels in samples collected during heat stress testing at 58 °C. Hollow bars indicate Salmonella levels in samples collected during heat stress testing at 48 °C. Similar (alphabetically adjacent) letters show no differences, and different letters show significant differences at α = 0.05.

The initial concentration of ST was 1.8 x 10⁶ CFU/mL in dairy wastewater. Overall, there was no significant interaction between temperature and time of sampling (p = 0.999) during the heat stress study. However, a significant reduction in ST levels was observed after the first 30 min incubation at 48 °C and 58 °C. The heat stress study showed that there was a 1.4 log₁₀ reduction from initial concentration after 30 min incubation at 48 °C, and a 2.5 log₁₀ reduction from initial concentration after 30 min incubation at 58 °C.

Figure 5 Changes in concentrations of Salmonella (average with standard error) in manure water in heat stress test at 48 °C and 58 °C.

After 30 min, the ST concentrations remained steady until 120 min and then there was significant difference in concentrations between the temperatures. During the time of heat stress testing, the reduction in ST concentration was more consistent and it went from 1.8 × 10⁶ CFU/mL at 0 min to 3.1 × 10² CFU/mL at 120 min at 58 °C. The results showed that the major die-off occurred during the initial phase of heating and within a short span of time at elevated temperature. Olsen and Larsen (1987) found that the time required for 90% decimation of Salmonella was 0.6 d to 0.9 d in anaerobic digestion of cattle slurry at 53 °C. Himathongkham et al. (1999) concluded that at lower temperatures (4 °C, 20 °C and 37 °C), the decimal reduction of Salmonella can be achieved in 65.8 d, 2.8 d and 2.5 d respectively.

During the last phase of the experiment perturbation of the ST was observed in both 48 °C and 58 °C, shown in Figure 6. Gray bars indicate Salmonella levels in samples collected from the study at 37 °C. Hollow bars indicate Salmonella levels in samples collected during heat stress test at 48 °C. Black bars indicate Salmonella levels in samples collected during heat stress test at 58 °C. Similar (alphabetically adjacent) letters show no differences, and different letters show significant differences at α = 0.05.

Figure 6 Concentrations of Salmonella typhimurium (average with standard error) at 37 °C, 48 °C and 58 °C in last 4 d of experiment. 

The concentrations of ST (on d 11) at 37 °C, 48 °C and 58 °C were 1.8 × 10⁶ CFU/mL, 4.8 × 10⁴ CFU/mL and 3.1 × 10² CFU/mL respectively. While comparing the three incubation temperatures (37 °C, 48 °C and 58 °C) during the last four days (d 11, d 12, d 13 and d 14) of the experiment; there was a significant interaction between temperature and day (p < 0.000 1).

The ST level was not detected in dairy wastewater on d 11 and d 12 at 48 °C and 58 °C. A sudden perturbation (i.e. increase in the concentrations of ST) was observed at both temperatures (48 °C and 58 °C) during d 13 and d 14, indicating a potential regrowth. Shepherd et al. (2010) evaluated the effect of heat shock (55 °C) on ST in dairy manure compost with vegetables under field conditions and found that heat stressed ST survived an additional 2 d during summer and 60 d during winter compared to non-heat shocked (30 °C) ST. Further studies are required to enhance the understanding of the regrowth of ST in dairy wastewater. While this study showed clearly that post treatment (after 10 d incubation at mesophilic temperature) of wastewater at elevated temperature (for 120 min) reduced the ST numbers considerably (~1.6 log₁₀ reduction at 48 °C and 3.8 log₁₀ reduction at 58 °C), and thus that the post treatment can be an effective method of controlling ST within a short period of time. Improved understanding is needed to reduce the risk of potential regrowth of ST in dairy wastewater.

The study was focused on assessing the impacts of temperature on ST inactivation due to the fact that temperature plays a crucial role in bacteria die-off. In addition to temperature, many other physical and chemical parameters (including change in pH, aerobic condition, anaerobic condition, mixing and non-mixing) control pathogen survival. Considering the enormous amount of dairy wastewater produced by the dairy industry, however, the use of chemical processes resulting in altering the pH is less likely to be an acceptable alternative for controlling pathogens in dairy manure. Further, changing the pH of dairy manure will require a large quantity of chemicals due to the high buffering capacity of manure (Angelidaki et al. 1993; Georgakakis et al. 1974).

Although, during the thermal inactivation, regrowth of Salmonella was observed at a later phase of the experiment (Figure 6), temperature driven inactivation is a viable and relatively simpler option for controlling the potential risk of foodborne pathogens such as Salmonella. Increased awareness of food borne pathogens and potential pathogen contamination caused by manure will likely to lead to limiting untreated manure application as fertilizer. If pathogen control restrictions are in place, the application of manure as fertilizer will largely depend on the availability of cost effective and simpler solutions for pathogen inactivation in manure.

Therefore identifying an improved method for pathogen control in manure is important. Pathogens such as Salmonella can seriously affect anyone, but for pregnant women and their fetuses, pathogens can be particularly harmful—even fatal (USFDA 2014b). Salmonella is the second most common cause of single etiology outbreaks and illnesses in the United States and Salmonella alone causes more than1.2 million infections each year, which account for $365 million in direct medical costs (CDC 2014). In 2012, Salmonella accounted for 25% of foodborne disease outbreaks, and 33% of illnesses which caused the most (64%) foodborne outbreak related hospitalizations in the United States (CDC 2014). It is not only a human health hazard but also responsible for clinical diseases in cattle like fever and diarrhoea (Smith 2002; Toth et al. 2011).

Many current manure treatment processes requires extensive time for reducing pathogens. Previous studies showed Salmonella survival in manure for extended periods of time. Toth et al. (2011) found survival of Salmonella enterica in dairy farm environments >137 d. Another study by Baloda et al. (2001) showed that Salmonella could survive ≤299 d. A study by Jones (1976) reported that Salmonella can survive in slurry or lagoon wastewater ≤286 d. Treatment methods such as the composting of dairy manure are used widely for treating dairy manure; however, the composting is also a slow process, and can be only applied for treating solid fractions of dairy manure. Currently, the U. S. Department of Agriculture (USDA) requires that there should be ≥120 d lag time between non-composted manure application and organic crop (with edible portions exposed to soil particles) harvesting (USDA 2000). While the existing USDA guidelines are useful for controlling the risk of pathogens in solid fractions of dairy manure, further science based information such as identifying the optimum retention period of dairy wastewater in storage lagoons can potentially assist in improving wastewater management. This study evaluated Salmonella inactivation in dairy manure under various thermal conditions; however, considering the growing number of Salmonella related food borne outbreaks and current ongoing food safety issues, additional studies focused on assessing Salmonella inactivation in both solid and liquid fractions of manure are required to improve manure management and its application as fertilizer.

4 Conclusions

The results of this study showed that there was a significant reduction in Salmonella concentrations over time particularly at elevated temperatures. The levels of Salmonella did not vary significantly within in the short period of time (i.e. ≈ 4 - 8 hours at mesophilic temperature). At elevated temperatures (thermophilic temperatures), the first 30 minutes of incubation was important for reducing Salmonella levels. The observed regrowth requires additional verification. Therefore, there is further scope of studies to understand the degradation patterns of Salmonella at thermophilic as well as mesophilic temperatures. The outcomes of the study will help in improving the understanding of Salmonella inactivation in dairy wastewater as well as deriving improved dairy wastewater management practices.  

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