Summary of key findingsDifferent groups of pathogens show different resistance to anaerobic digestion and its operational conditions (e.g. T and pH). While higher temperatures generally lead to higher pathogen reductions, the reactor design, feeding strategy or hydraulic retention time did not have a significant effect. Other than environmental parameters, biological activity seems to have a crucial role in pathogen reduction.Background and relevanceAnaerobic digestion (AD) is a well stablished technology to treat organic waste, allowing energy recovery in the form of biogas and nutrient as well as carbon recovery via digestate utilization (Capson-Tojo et al., 2016). The high contents of nutrients (and C) in digestate make it a notable fertilizer and/or soil improver (Guilayn et al., 2020). However, this practice is not free of concerns, since agricultural digestate use can contribute to pathogen dissemination into the environment if it is not managed properly (Ma et al., 2022). To prevent and minimize risks to human, animal and environmental health, the European Union has provided guides for standard practices and protocols for operating AD plants (i.e. EC2019/1009 or EC142/2011). Sampling collection protocols and microbiological standards were also included in the regulation, ensuring a safe digestate use. Fulfilling the concentration limits of targeted pathogens is therefore crucial, as their presence might limit the application of the digestate.Pathogen inactivation during AD has been widely studied over the past decade. However, most studies have focused on a particular pathogen and/or specific operational conditions. Thus, a more global view of the process is needed to identify the main operational conditions affecting AD hygienization efficiency. This literature review aims at reunite the available experimental-data to increase our understanding on the AD capacity for pathogen inactivation. Particular attention was paid to the impact of operational conditions and reactor design/type, as conflicting results exist. This study could potentially contribute to the definition of new standard protocols and operational guides, aiming at enhancing the hygienization capacity of AD.Materials and MethodThe bibliographic survey was performed with Web of Science (Clarivate) in October 2022. A total of XXX studies were initially screened. Omission of studies from this analysis was done on the basis of missing main inputs and/or outputs, reported units not allowing calculating pathogen reduction, or inconsistent data. Finally, quantitative and qualitative data were extracted from 68 studies, obtaining a database consisting of 1246 observations. Each observation included relevant information about a particular experiment (e.g. AD conditions, inoculum, reactor design, substrate fed, or main process outcomes, such as methane yield or biogas productivity). Plots and statistical analyses were performed using the software R 4.1.2. ANOVA tests were performed using a significance threshold of p = 0.05.Results and discussionThe first result to be outlined from the meta-analysis performed is the confirmation of the generally well-accepted relative resistance that pathogens have during AD. Median overall reductions in pathogen concentrations during AD (input vs. output) were more significant for Gram negative bacteria, followed by viruses, viable eggs (of nematodes), Gram positive bacteria, and spore-forming bacteria, the latest being those most resistant to AD (dedicated figure not shown; see Figures 1.1 and 1.2). These different reduction values are related to well-known survival mechanisms and adaptive traits that pathogens possess. See Lin et al. (2022) for a deeper discussion.Figure 1.1 shows that temperature did not affect equally the different pathogen groups identified. Most of observations targeted Gram negative bacteria (n=671), compared to Gram positive bacteria (n=86) or viruses (n=40). While Gram negative bacteria, and to a lesser extent Gram positive bacteria, were considerably affected by temperature (higher values lead to higher reductions), the reduction in spore-formers was not significantly increased until temperatures over 50ºC were reached. No effect of temperature on reduction in viruses could be determined. Regarding pH values (Figure 1.2), the overall reduction extent for each pathogen group was similar to these values at different temperatures, but the impact of pH on each pathogen differed. While the reduction of Gram negative bacteria was higher at both low (< 6.5) and high (> 8.5) pH values, the reduction of Gram positive bacteria was higher at decreasing pH values, and that of spore-formers and viruses increased with pH values. The different behaviors for each pathogen group are explained by their physiology and their survival mechanisms. The results for both temperature and pH were confirmed by analyses focused on common representative organisms for each pathogen group, commonly targeted by legislations (i.e. Escherichia coli, Enterococcus spp., Clostridium perfringens and Ascaris suum).Figure 1.3 shows the overall pathogen reductions in reactors operated at different feeding modes (e.g. batch, semi-continuous and continuous) and at different scales (e.g. laboratory and pilot). No significant differences were found in the laboratory-scale reactors operated at different feeding modes, suggesting that the feeding strategy does not impact pathogen removal (most data points came from laboratory scale studies). Interestingly, the available data show that the obtained pathogen reductions are not affected by the reactor scale, suggesting that laboratory-scale data can be extrapolated safely to larger scale installations (pathogen reductions can even be higher at pilot scale; see Figure 1.3).Results from different reactor designs (Figure 1.4) further confirm that the feeding strategy does not affect pathogen reduction. Similarly, overall reductions for different reactor designs were similar for most cases. Although there is little research yet (n=58), it seems that temperature phased anaerobic reactor (TPAD) is a process configuration that seem to provide higher reductions. This is mostly due to the higher temperatures in the 1st stage (generally thermophilic), as the reductions in the 2nd stage (generally mesophilic) is insignificant (see Figure 1.4). Thermophilic temperatures (~50ºC) result in higher reductions (Figure 1.1), explaining the higher apparent performances of TPADs. Indeed, thermophilic stirred-tank reactors (STRs) resulted in similar reductions (figure not shown). The higher reductions in 1st stages also suggest that long hydraulic retention times (HRTs) might not be required. The collected data from continuous and semi-continuous reactors showed that higher HRTs over XX days did not lead to higher overall pathogen reductions (figure not shown). Tests with different pathogen groups and representative organisms further confirmed this result. This is obviously interesting from a design point of view, as HRTs do not need to be increased to improve pathogen reductions. Conversely, the duration of the experiment during batch test did have a significant impact on pathogen removal (particularly in the fisrt 20-30 days; results not shown). This observation could not be drawn from comparing the overall abatements between feeding modes (Figure 1.3) due to large deviations and similar median/mean performances. The significant impact of the batch duration suggests that the microbial activity plays a main role during hygienization, and not only the operational conditions (e.g. T and pH). As for the HRT, the organic loading rates did not affect the obtained pathogen reductions. Regarding process outputs, neither the methane yields nor the productivities impacted the resulting pathogen reductions. This implies that optimal conditions should not be related with change in pathogen reduction. Concerning the hygienization capacity of VFAs in the reactors, we cannot make clear statements, as most data points were obtained at low total VFA concentrations below 0.5-1.0 g COD·L-1.These observations shed light on the overall effect of operational conditions and reactor design/type on the removal of the hygienization performance for different pathogens. This might serve to further understand the process and to develop optimized operational strategies tailored to each situation, aiming at maximizing resource recovery by allowing a safe application of digestate streams. This literature review highlighted the need to acquire new knowledge about the impact of different pretreatment and post-treatment approaches on hygienization performance.ReferencesCapson-Tojo, G., Rouez, M., Crest, M., Steyer, J.-P., Delgenès, J.-P., Escudié, R. (2016). Food waste valorization via anaerobic processes: a review. Rev. Environ. Sci. Bio/Technology 15, 499–547.Guilayn, F., Rouez, M., Crest, M., Patureau, D., Jimenez, J. (2020). Valorization of digestates from urban or centralized biogas plants: a critical review, Rev. Environ. Sci. Bio/Technology 19, 419-462.Lin, M., Wang, A., Ren, L., Qiao, W. (2022). Challenges of pathogen inactivation in animal manure through anaerobic digestion : a short review. Bioengineered 13, 1149–1161.Ma, G., Chen, Y., Ndegwa, P. (2022). Anaerobic digestion process deactivates major pathogens in biowaste : A meta-analysis. Renew. Sustain. Energy Rev. 153, 111752.