Persistence and Potential Viable but Non-culturable State of Pathogenic Bacteria during Storage of Digestates from Agricultural Biogas Plants

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 NH4+/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.

Ruminant and chicken: important sources of campylobacteriosis in France despite a variation of source attribution in 2009 and 2015

“…As previously suggested 19 , 33 , 35 , contact with animals and the environmental contamination by ruminants, including water contamination, need also to be considered since Campylobacter spp. were shown to survive in bovine manure 53 or during anaerobic digestion of livestock effluents in biogas plant 54 , 55 . Waterborne transmission of Campylobacter from ruminant to human has been previously reported 56 and a recent 2-year study highlighted a high prevalence (80.7%) of Campylobacter spp.…”

Antibiotic Use in Agriculture and Its Consequential Resistance in Environmental Sources: Potential Public Health Implications

“…The authors suggested that the rate of survival of these bacteria depended on the temperature of the operating process in association with the duration of the fermentation process and the microbial composition. Ostensibly, Maynaud et al [ 214 ] further confirmed the occurrence of enteric pathogenic bacteria in the digestate obtained from the anaerobic biodigester. The authors emphasized the potential of the viable, but non-cultural state of bacteria, which might cause the regrowth of pathogens during digestate storage, prior to land spreading.…”

Sources and survival ofListeria monocytogeneson fresh, leafy produce

“…; Maynaud et al . ). Given that the soil environment may result in nutrient deprivation and other stresses known to induce VBNC, this may also cause L. monocytogenes to turn VBNC, but data on this characteristic of the bacterium in the soil environment is missing.…”

“…A variety of pathogenic bacteria including L. monocytogenes enter a protective VBNC state in response to nutrient starvation, incubation outside the normal temperature, increased or reduced osmotic concentrations and heavy metal exposure (Oliver 2010). Indeed, research has shown that a large fraction of the L. monocytogenes population becomes VBNC in microcosms containing pig manure and digestates from agricultural biogas plants (Desneux et al 2016;Maynaud et al 2016). Given that the soil environment may result in nutrient deprivation and other stresses known to induce VBNC, this may also cause L. monocytogenes to turn VBNC, but data on this characteristic of the bacterium in the soil environment is missing.…”