Livestock waste, food waste and sewage sludge are common organic wastes in Ireland. All of them are rich in organic carbon and nutrients like nitrogen, phosphorus and potassium - but they are also reservoirs of various pathogenic microorganisms, write Yan Jiang and Xinmin Zhan of NUI Galway.

Organic wastes and viruses

Livestock waste, food waste and sewage sludge are common organic wastes in Ireland. All of them are rich in organic carbon and nutrients like nitrogen, phosphorus and potassium.

It thereby makes anaerobic digestion and composting prevalent treatment methods for recovery of methane-rich biogas and nutrient-rich fertiliser. However, these organic wastes are also reservoirs of various pathogenic micro-organisms.

The commonly concerned pathogens in these organic wastes include Gram-positive and Gram-negative bacteria, viruses, protozoa and nematode (Jiang et al., 2020). If not managed properly, pathogens may infect human beings and animals, causing serious public health problems.

Viruses usually exist at high concentrations in organic wastes, and are persistent in the environment. The types of viruses in different organic wastes vary.

For instance, the most prevalent viruses in dairy manure include coronavirus, enterovirus, polyomavirus, adenovirus and rotavirus (Burch et al., 2018), influenza, noroviruses, sapoviruses, hepatitis E virus, parvovirus and rotavirus in swine manure (Ziemer et al., 2010). World Health Organization (WHO) and Foodborne Diseases Active Surveillance Network (FoodNet) report that the main foodborne viruses are adenovirus, reovirus, bacteriophages, norovirus and hepatitis A virus (Magri et al., 2015; WHO, 2015).

The commonly detected viruses in sewage sludge include norovirus, rotavirus, adenovirus and hepatitis E virus (El-Senousy & Abou-Elela, 2017).

Viruses do not have a cellular structure. Virion, existing as a virus particle, consists of a nucleic acid core, a protective protein coating called capsid, and some cases an outside lipid envelope derived from the host cell.

With different genetic materials (DNA or RNA), viruses are divided into four types: single- and double-stranded DNA (ssDNA and dsDNA), and single- and double-stranded RNA (ssRNA and dsRNA). Thus the inactivation of viruses can be related to DNA/RNA cleavage, capsid protein denaturation and envelop damage.

Coronaviruses are ssRNA viruses with envelopes, which can cause gastrointestinal, respiratory, nervous and mammary gland diseases. Infections caused by rare species can be lethal, such as SARS and COVID-19.

Several coronaviruses have been identified in livestock manure, for example, porcine and bovine respiratory coronaviruses, transmissible gastroenteritis virus, bovine enteropathogenic coronavirus, porcine hemagglutinating encephalomyelitis virus, porcine deltacoronavirus, porcine epidemic diarrhea virus, and a non-defined bat-HKU2-like alphacoronavirus (Boileau & Kapil, 2010; Saif et al., 2019). Chinese researchers have found COVID-19 in feces of the patients.

Hence workers must wear gloves, masks and other personal protective equipment (PPE) when working with organic wastes during collection, transportation, treatment, etc.

Inactivation of viruses during anaerobic digestion

The inactivation mechanisms involved in anaerobic digestion include temperature, volatile fatty acids (VFAs) and ammonia. Anaerobic digestion is usually operated at mesophilic (35 ℃) or thermophilic (55 ℃) temperatures.

As a Category 3 animal by product (ABP), livestock manure was initially stipulated to be thermal treated at 70 ℃ for 60 min (EC No. 1774/2006). A further amending regulation (EC No. 208/2006) indicated that other treatment processes may be used if the validation was demonstrated to cause at least 3 log₁₀ reductions on viruses.

Four stages are included in aerobic digestion: hydrolysis, acidification, acetogenesis and methanogenesis. VFAs are main intermediate products during anaerobic digestion, and the concentrations could be extremely high by hydrolysis and acidification of high organic matter wastes, e.g. food waste. Ammonia, produced from the degradation of protein, is naturally present at high level in livestock manure.

At ambient and mesophilic temperatures, the inactivation of viruses is related to genome types. Viruses with ssRNA are most labile, with the inactivation rates 2-3 orders of magnitude greater than those of the other genome types (Decrey et al., 2016). DNA and dsRNA viruses have more stable nucleic acid, and the inactivation is mainly attributed to the degradation of viral proteins.

It explains why dsRNA and DNA viruses, e.g. adenovirus, polyomavirus and rotavirus, are resistant and frequently detected during livestock manure storage and mesophilic digestion, while ssRNA viruses, e.g. hepatitis A/E virus, norovirus, enterovirus and astrovirus, are rarely detected. While at thermophilic temperatures, the gaps between inactivation rates of different genome types narrowed.

Increase of temperature causes exponential damage to viruses. As the temperature increases from 20 to 60 ℃, the inactivation rates of bacteriophages MS2, ΦX174 and T4 increases from about 10^-2 day^-1 to as high as 10²-10³ day^-1 (Decrey et al., 2016).

In livestock slurry, the inactivation times of some viruses (including classical swine fever virus, swine influenza virus, Aujeszky’s disease virus, food and mouth disease virus, bovine diarrhea virus and rhinotracheitis virus) are 3-24 h at 35 ℃, and 5-60 min at 55 ℃ (Haas et al., 1995). Emmoth (2010) reported that the log₁₀ reductions of 1.1 and 2.6 for porcine parvovirus at 55 and 70 ℃ for 60 min, respectively.

Ammonia is toxicity to viruses. The presence of ammonia at 20-40 mmol/L (300 – 600 mg/L) results in a 100-1000 fold increase in the inactivation rate of ssRNA viruses (Decrey et al., 2016). Ammonia can also increase the inactivation rates of DNA and dsRNA viruses, but not as dramatic as ssRNA ones.

The effect of VFAs on virus inactivation is rarely reported. Our group studied inactivation of pathogens during mesophilic dry co-digestion of pig manure and food waste.

The results showed that total coliforms and E. coli were reduced to below the detection limit within 4-7 days; Salmonella was totally eliminated with seven days; enterococci was more resistant, but was inactivated in 12-26 days (Jiang et al., 2018a; Jiang et al., 2018b).

The high VFA accumulation was the main inactivation factor for their inactivation. Because these bacteria can act as hosts of viruses, we consider the accumulation of VFAs during anaerobic digestion can contribute to virus inactivation as well.

The real inactivation rates of viruses in full-scale digesters are combined effects of temperature, VFAs and ammonia. Survey on seven full-scale mesophilic anaerobic digestion plants treating dairy manure indicated that the maximum log₁₀ reductions of viruses (including bovine polyomavirus, group A rotavirus, bovine enterovirus, bovine coronavirus and bovine adenovirus) ranged from 0.51 to >2.00 (Burch et al., 2018).

The inactivation rates were less than a half of the values reported in literature. For widely used mesophilic AD projects, a pasteurisation process is highly recommended before land application or other reuse of the digestate

Inactivation of viruses during composting

The inactivation of pathogens during composting is mainly attributed to the thermal effect. During composting, organic matters are decomposed into humus-like materials by aerobic microorganisms at the presence of oxygen, and the temperature can be maintained at as high as 55-70 ℃ for three to seven days, or even longer.

The high temperature during composting can inactivate most viruses to get hygiene safe compost. Analysis of 594 samples from different stages in five full-scale pig manure composting plants showed that hepatitis E virus (ssRNA) was not detected in any compost samples (Garcia et al., 2014). It indicates the effectiveness of composting for virus elimination.

Inaction of coronavirus

Coronaviruses are labile at ambient or higher temperatures. The transmissible gastroenteritis virus in pig slurry can live for 2 weeks, 24 h and 30 min at 20, 35 and 55 °C, respectively (Haas et al., 1995).

Bovine coronavirus was not detected in the effluent of seven full scale mesophilic anaerobic digestion plants treating dairy manure, indicating mesophilic anaerobic digestion can inactivate coronavirus effectively (Burch et al., 2018).

Coronavirus is also sensitive to ammonia. Feline coronavirus can be reduced by ≥ 5.0 log₁₀ reductions at the ammonia concentration of 121 mmol/L (c.a. 2000 mg/l) within four hours (Emmoth, 2010). Anaerobic digestion and composting should be effective in coronavirus inactivation.

Conclusion

The inactivation mechanisms of viruses in anaerobic digestion involves temperature, VFAs and ammonia, while in composting thermophilic temperature is the main inactivation factor.

During thermophilic anaerobic digestion and composting, most viruses can be inactivated due to the thermal effect. In mesophilic anaerobic digestion, labile ssRNA viruses (including coronavirus) can be inactivated effectively.

Dry anaerobic digestion, which can accumulate high VFA and ammonia concentrations, may provide a relative high-effective way for pathogen inactivation.

References

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2.) Burch, T.R., Spencer, S.K., Borchardt, S.S., Larson, R.A., Borchardt, M.A. 2018. Fate of Manure-Borne Pathogens during Anaerobic Digestion and Solids Separation. J Environ Qual, 47(1), 336-344.

3.) Decrey, L., Kazama, S., Kohn, T. 2016. Ammonia as an In Situ Sanitizer: Influence of Virus Genome Type on Inactivation. Appl Environ Microbiol, 82(16), 4909-20.

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