Global demand for fertilizer nutrients, including nitrogen, phosphorus, and potassium, is expected to reach 200,500,000 tons by 2018, and food demand for human consumption and livestock feed is expected to increase by 60–110% by 2050. Food production sustainability is therefore essential, and will require nutrient and water recycling under appropriate conditions of health and safety (Tilman et al., 2001; Food and Agriculture Organization of the United Nations [FAO], 2014). Swine manure is biomass suitable for biogas production in anaerobic biodigesters (AB) and the final digestate is used as agricultural biofertilizer. Digestate recycling is ecologically and economically viable, because it can simultaneously biofertilize and irrigate various crops, such as maize, wheat, and soybean, satisfying some or even all nitrogen and phosphorus requirements (Hansen et al., 1998; Topp et al., 2009).

Human and animal diseases associated with soil can be caused by exposure to or ingestion of indigenous pathogens of the normal soil microbiota, food contaminated with enterotoxins and neurotoxins, or enteric pathogens from human or animal excreta (Santamaría and Toranzos, 2003). Swine digestate can contain high levels of enteric pathogens that may contaminate soil, water, and foods (Khetsuriani et al., 2006; Topp et al., 2009). Its use in agriculture is therefore an issue for global “One Health” as it may affect humans and animals and, indeed, the environment more generally. The enteric pathogens present in biofertilizers can accumulate in soil (Gerba and Smith, 2005). Microbial models can be used to assess the behavior of enteric pathogens, including their survival, flow, propagation, leaching, percolation, and inactivation (Langlet et al., 2008; Boudaud et al., 2012). Bacteriophages, such as somatic coliphages (e.g., PhiX-174), f-specific RNA phages (e.g., MS2), and mengovirus (vMC₀) have been used as models for human and animal enteric viruses (Langlet et al., 2008; Boudaud et al., 2012). Similarly, enteric bacteria, particularly coliform and Salmonella ssp., are widely used as biomarkers of fecal contamination. Salmonella is one of the most prevalent bacterial pathogens; it is zoonotic and has high survival rates in environment (Griffith et al., 2006; Venglovsky et al., 2006; European Food Safety Authority, and European Centre for Disease Prevention, and Control, 2015).

There have been few rigorous analyses of how enteric microorganisms behave in soil after being introduced by biofertilization, or of rates of environmental survival and propagation, leaching and percolation in different types of soil. Such analyses are required before swine-derived biofertilizers can be widely exploited agriculturally. The movement of enteric bacteria through soils, and the percolation of microorganisms in soils more generally, depend largely on the water saturation state (Artz et al., 2005). European Regulation (EC) 1069/2009, addressing health aspects of animal by-products and derived products not intended for human consumption, requires that Salmonella spp. to be below the detection threshold (25–50 g samples of biofertilizer scoring negative) and fewer than 1000 E. coli CFU g^-1 of wet weight of biofertilizer (EC, 2009). In some countries, for example Brazil, there are no specific domestic or international rules for recycling animal by-products (Kunz et al., 2009). There is an evident need for the rigorous assessment of the fate of important enteric microorganisms after biofertilization of different types of soils. The development of appropriate bio-models for diverse environmental scenarios (survival, percolation, and leaching) would be valuable. However, there are currently no reports in the literature suggesting models that could be used to investigate the stability, percolation, and leaching of enteric pathogens, simultaneously. There has also been no comparison of the behaviors of enteric viruses and bacteria in sandy and clay soils in ex situ studies.

We aimed to (i) evaluate the survival and percolation of several major enteric microorganisms and to (ii) select the most suitable enteric microorganisms to serve as biomarkers of percolation and leaching after biofertilization with swine digestate. We spiked swine digestate with several model microorganisms (PhiX-174, vMC₀, Salmonella enterica Typhimurium and E. coli O157:H7) and applied samples to clay and sandy soils. We then evaluated survival, percolation and leaching of the added microorganisms.

Soil features, including temperature, pH, clay, organic matter, and salinity content, affect the survival, stability, percolation, and leaching of enteric pathogens present (Kimura et al., 2008). We investigated the survival and percolation of four relevant enteric microorganisms (two bacterial pathogens: E. coli O157:H7, and S. enterica Typhimurium and two viruses: PhiX-174, vMC₀) in clay and sandy soils in a model of biofertilization with swine effluent from AB. E. coli O157:H7 and Salmonella ssp. are major zoonotic enteric bacterial pathogens. Salmonella ssp. are often isolated from farmed pigs and poultry, which are the main reservoirs for human infections. These species show high survival rates in the environment (Griffith et al., 2006; Chen and Jiang, 2014). Somatic coliphages and vMC₀ have been used as models for human and animal enteric viruses. PhiX-174 is stable across a range of temperatures, pHs and UV treatments, and vMC₀ has been used as model of norovirus, and hepatitis A and E viruses (Costafreda et al., 2006; Rodríguez-Lázaro et al., 2012; Boudaud et al., 2012).

Soils are complex matrixes and some of the compounds present have antimicrobial (virucidal and/or bactericidal) properties when concentrated: examples include malic, lactic and butyric acids, proteases, nucleases, bacterial and fungal metabolites; there may also be microorganism predation (Wiesmann et al., 2007; Dika et al., 2011). The survival, percolation, and leaching of the four microorganisms we studied differed between the two soil types. However, microbial survival and leaching was significantly lower in clay soil than in sandy soil for all the four microorganisms. The organic matter and nutrient contents are higher in clay than sandy soil, and the pH more alkaline. The pH and salt concentration (salt increases conductivity) may directly affect the adsorption capacity of viruses. Increasing the concentration of cations, especially multivalent cations, increases the adsorption capacity, mainly for enteric viruses (Gerba, 1984; Gerba and Smith, 2005; Pepper et al., 2006). The leaching of nutrients is high on sandy soils with poor water retention capacity (Department for Environment, Food and Rural Affairs, 2010). PhiX-174 showed faster percolation and leaching from biofertilized sandy soil than clay soil. The isoeletric point of PhiX-174 is 6.5 (Schijven and Hassanizadeh, 2000) and the pH of clay and sandy soils studied was 8.6 and 6.7, respectively. Therefore, PhiX-174 may have aggregated in the clay soil, hindering its percolation and leaching, whereas this was unlikely to have been the cases in sandy soil.

We report that the survival of the model viruses was significantly longer and correspondingly that microbial inactivation was faster for bacteria (iT₉₀ days). Bacteriophage PhiX-174 showed the least inactivation the highest iT₉₀ value of the four microorganisms studied (Table 2 and Figure 1). PhiX-174 may therefore be useful as a biomarker for the study of persistence of enteric pathogens in soils. Enteric viruses can survive for long periods [DNA viruses like human adenovirus decay by only 90% in 180 days in biosolid-amended and un-amended soils (Schwarz et al., 2014)] particularly in soils with a higher moisture content (60–90 days in soils with 10% moisture content and 15–25 days in air-dried soil (Yeager and O’Brien, 1979).

All four microorganisms percolated significantly in both soil types. Thus, a proportion of the microorganisms applied at the surface can be transferred into deeper layers, and the reduction of microbial survival may be, in part, associated to this process. The percolation of the microorganisms was linear, but microbial accumulation in the deeper layers was logarithmic. S. enterica Typhimurium percolated and was inactivated more slowly than E. coli O157:H7, suggesting that it may readily remain in contact with vegetables of short roots, like lettuce. E. coli O157:H7 was both transferred to the deeper layers of both soils (i.e., percolation) and leached faster, raising the issue of contamination of surface and groundwater used for human supply and for plant irrigation. E. coli O157:H7 and Salmonella serovar Typhimurium are able to move through soil with water after rainfall or irrigation and can even reach the groundwater (Artz et al., 2005; Lang and Smith, 2007; Semenov et al., 2009). Field experiments showed that 20% of E. coli applied to fields in contaminated slurry was found in drain water (Vinten et al., 2002). Also, our study and previous results reveal the risk of internal contamination of plants by E. coli O157:H7 and other coliforms. In view of these observations, international regulations are surprisingly divergent about microbiological criteria for water for human consumption and irrigation (Chart, 2000; Semenov et al., 2009).

We show that E. coli O157:H7 has potential as a microbial biomarker for depth contamination and leaching in clay and sandy soils. Note that it is possible that genogroups of E. coli different from E. coli O157:H7 behave differently in these or other soils.

We report that survival, percolation, and leaching of enteric microorganisms in biofertilized soils depend on the characteristics of the microbes and the soil. Our findings corroborate previous work suggesting that pH, OM content, soil texture, and rainfall are the principal factors that affect the survival and leaching of microbial pathogens (Demoling et al., 2007). We believe that potential microbial contamination of surface and groundwater by leaching and percolation should be evaluated prior to swine digestate application in agriculture. Finally, our work shows that the bacteriophage PhiX-174 and the bacterial pathogen E. coli O157:H7 can be used as biomarkers for studies on microbial persistence and percolation and leaching, respectively. Our findings contribute to the development of predictive models of the behavior of enteric pathogens in different soils and of the potential transfer of pathogens to water and food by biofertilization. Such models would be useful for risk management and mitigation in swine digestate recycling.

GF performed most of the experiments and help in the preparation of the manuscript. MG-G discussed the experiments, review the results and help in the preparation of the manuscript. MH discussed the experiments, supervise the experimental work and review the results and help in the preparation of the manuscript. AK review the experimental plan and the results. CB discussed the experiments, review the results and help in the preparation of the manuscript. DR-L designed the experiments, review the results, and prepare the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.