Efficacy of NH3 as a secondary barrier treatment for inactivation of Salmonella Typhimurium and methicillin-resistant Staphylococcus aureus in digestate of animal carcasses: Proof-of-concept

Abstract

Managing the disposal of infectious animal carcasses from routine and catastrophic disease outbreaks is a global concern. Recent research suggests that burial in lined and aerated trenches provides the rapid pathogen containment provided by burial, while reducing air and water pollution potential and the length of time that land is taken out of agricultural production. Survival of pathogens in the digestate remains a concern, however. A potential answer is a 'dual'-barrier approach in which ammonia is used as a secondary barrier treatment to reduce the risk of pathogen contamination when trench liners ultimately leak. Results of this study showed that the minimum inhibitory concentration (MIC) of NH3 is 0.1 M (~1,468 NH3-N mg/L), and 0.5 M NH3 (~7,340 NH3-N mg/L) for ST4232 & MRSA43300, respectively at 24 h and pH = 9±0.1 and inactivation was increased by increasing NH3 concentration and/or treatment time. Results for digestate treated with NH3 were consistent with the MICs, and both pathogens were completely inactivated within 24 h.

Fig 1. Overall experimental design including primary and secondary barrier treatments.

Fig 2. The effect of ammonia on inactivation of Salmonella Typhimurium χ4232 in phosphate buffer solution (PBS).

Note: pH = 9.0±0.1, T = 35°C. NH₃ concentrations of 0.052 M, 0.105 M, 0.315 M, and 0.524 M are equivalent to 734, 1,468, 4,404, and 7,340 NH₃-N mg/L, respectively (estimated mole fraction of NH₃-N to TAN = 0.529). Source of NH₃ was (NH₄)₂SO₄. Different letters within each treatment time indicate significant difference (p<0.05), n = 3.

Fig 3. The effect of ammonia on inactivation of methicillin resistant Staphylococcus aureus MRSA ATCC 43300 in phosphate buffer solution (PBS).

Note: pH = 9.0±0.1, T = 35°C. NH₃ concentrations of 0.052 M, 0.105 M, 0.315 M, and 0.524 M are equivalent to 734, 1,468, 4,404, and 7,340 NH₃-N mg/L, respectively (estimated mole fraction of NH₃-N to TAN = 0.529). Source of NH₃ was (NH₄)₂SO₄. Different letters in each treatment time indicate significant difference (p<0.05), n = 3.

Fig 4. The effect of MIC of ammonia (0.105 M NH₃; the equivalent of 1,468 NH₃-N mg/L) on inactivation of ST4232 in digestate of week #3 of poultry carcass aerobic digestion.

Source of NH₃ was (NH₄)₂SO₄. The control contained only residual TAN (0.039 M) in the digestate collected at week #3 [24]. Week #3 represents an early-phase of decomposition characterized by high BOD, highest TSS & VSS levels, and the first visual evidence of the whole carcass breakup and disintegration. Data represent the means log₁₀ of measured Salmonella concentrations of 4 aerobic reactors ± SD. Different letters in each treatment time indicate significant difference (p<0.05), n = 3.

Fig 5. The effect of MIC of ammonia (0.105 and 0.524 M NH₃; the equivalent of 1,468 and 7,340 NH₃-N mg/L) on the survival of newly seeded ST4232 in digestate of week #13 poultry carcass aerobic digestion.

Source of NH₃ was (NH₄)₂SO₄. The control contained only residual TAN (0.00057 M) in the digestate collected at week #13 [24]. Week #13 represents a late-phase of a complete aerobic digestion represented by lower microbial activity post the 99% reduction of BOD, TSS, and VSS. Data represents the mean log₁₀ of measured Salmonella concentrations of 4 aerobic reactors ± SD. Different letters in each treatment time indicate significant difference (p<0.05), n = 3.

Fig 6. The effect of MIC of ammonia (0.105 and 0.524 M NH₃; the equivalent of 1,468 and 7,340 NH₃-N mg/L) on the survival of newly seeded MRSA43300 in digestate of week #13 poultry carcass aerobic decomposition.

Source of NH₃ was (NH₄)₂SO₄. Week #13 represents a late-phase of a complete aerobic digestion represented by lower microbial activity post the 99% reduction of BOD, TSS, and VSS. Data represents the mean log₁₀ of measured concentrations of MRSA43300 of 4 aerobic reactors ± SD. Different letters in each treatment time indicate significant difference (p<0.05), n = 3.