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Abstract
Despite the development of on-farm anaerobic digestion as a process for making profitable use of animal by-products, factors leading to the inactivation of pathogenic bacteria during storage of digestates remain poorly described. Here, a microcosm approach was used to evaluate the persistence of three pathogenic bacteria (Salmonella enterica Derby, Campylobacter coli and Listeria monocytogenes) in digestates from farms, stored for later land spreading. Nine samples, including raw digestates, liquid fractions of digestate and composted digestates, were inoculated with each pathogen and maintained for 40 days at 24°C. Concentrations of pathogens were monitored using culture and qPCR methods. The persistence of L. monocytogenes, detected up to 20 days after inoculation, was higher than that of Salmonella Derby, detected for 7–20 days, and of C. coli (not detected after 7 days). In some digestates, the concentration of the pathogens by qPCR assay was several orders of magnitude higher than the concentration of culturable cells, suggesting a potential loss of culturability and induction of Viable but Non-Culturable (VBNC) state. The potential VBNC state which was generally not observed in the same digestate for the three pathogens, occurred more frequently for C. coli and L. monocytogenes than for Salmonella Derby. Composting a digestate reduced the persistence of seeded L. monocytogenes but promoted the maintenance of Salmonella Derby. The effect of NH/NH3 on the culturability of C. coli and Salmonella Derby was also shown. The loss of culturability may be the underlying mechanism for the regrowth of pathogens. We have also demonstrated the importance of using molecular tools to monitor pathogens in environmental samples since culture methods may underestimate cell concentration. Our results underline the importance of considering VBNC cells when evaluating the sanitary effect of an anaerobic digestion process and the persistence of pathogens during the storage of digestates and subsequent land spreading.
Highlights
Anaerobic digestion (AD) is one option for managing organic waste originating from agriculture, industry or wastewater treatment
In order to improve risk assessment of animal by-products when re-used in agriculture, we investigated the persistence during the storage of digestates of three pathogenic bacteria found in animal feces: Salmonella Derby (Huong et al, 2014), Campylobacter coli (Schweitzer et al, 2011; Egger et al, 2012) and Listeria monocytogenes (Desneux et al, 2016)
The nine digestates spiked with cells of L. monocytogenes, Salmonella Derby and C. coli were monitored for 40 days by both the culture method and quantitative PCR (qPCR) assay
Summary
Anaerobic digestion (AD) is one option for managing organic waste originating from agriculture, industry or wastewater treatment. The use of digestate as fertilizer inevitably involves the issue of health risks associated with pathogenic microorganisms present in waste of fecal origin (such as manure, slurry or sludge): such pathogens may be spread along with the digestate on agricultural soils (Smith et al, 2004; Goberna et al, 2011; Johansen et al, 2013), with consequent contamination of the food chain Among them, these pathogens number a variety of viruses, including swine hepatitis E virus, bacteria including Salmonella sp., Listeria monocytogenes, Mycobacterium paratuberculosis, thermotolerant Campylobacter and parasites including Cryptosporidium parvum and Giardia sp. From 2008 to 2014, salmonellosis decreased by 44%, mainly due to the successful Salmonella control programs
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